Multiplex Y-STR analysis

ABSTRACT

Novel Y-STR multiplex analysis designs, primer design, allelic ladders, methods of use and kits are disclosed, including the use of primer sets designed to provide amplicons for at least 11 Y-STR loci having a base pair size of less than about 220 bp, as well as the use of primer sets designed to provide amplicons for at least 22 Y-STR loci including at least 5 rapidly mutating loci.

This application is a divisional of U.S. patent application Ser. No.13/828,443 filed Mar. 14, 2013, and claims the benefit of priority under35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/697,742 filedSep. 6, 2012; U.S. Provisional Application No. 61/720,949 filed Oct. 31,2012; U.S. Provisional Application No. 61/761,152 filed Feb. 5, 2013;and U.S. Provisional Application No. 61/765,323 filed Feb. 15, 2013;each of which disclosures is herein incorporated by reference in itsentirety.

The section headings used herein are for organizational purposes onlyand should not be construed as limiting the subject matter describedherein in any way.

FIELD

In general, the disclosed invention relates to the determination of theidentity of short tandem repeat (STR) alleles on the Y chromosome of ahuman using a multiplex analysis process. A multiplex analysis thatincludes increased numbers of loci that can provide increaseddiscrimination and sensitivity may accurately genotype a wider range ofindividuals.

BACKGROUND

The fields of forensics, paternity testing, cell line ID, andpersonalized medicine routinely use DNA-based techniques for identitydeterminations, genotyping, phenotypic prediction, and in the predictionand/or prevention of disease. DNA typing involves the analysis of selectregions of genomic DNA, commonly referred to as “markers.” Most typingmethods in use today are specifically designed to detect and analyzedifferences in the length and/or sequence of one or more regions of DNAmarkers known to appear in at least two different forms in a population.Such length and/or sequence variation is referred to as “polymorphism.”Any region (i.e., “locus”) of DNA in which such a variation occurs isreferred to as a “polymorphic locus.”

In recent years, the discovery and development of polymorphic shorttandem repeats (STRs) as genetic markers has played an important role inDNA typing. STRs have become the primary means for human identity andforensic DNA testing.

In particular, Y-STR analysis is a valuable tool in a number ofapplications. Forensic applications include use in investigation ofsexual assault cases where male DNA may be present in a sample that alsocontains an excess of female DNA. Y-STR analysis can be critical inexcluding individuals from further inquiries. In another forensicapplication, a sample may include DNA from multiple male contributors.Y-STR analysis can be used to trace family relationship among males,either in forensic or other inheritance analyses, and can be used inmissing person investigations. Additionally, Y-STR analysis can be usedin paternity testing, including scenarios where the alleged father isnot available for direct comparison.

One database used to assist investigators is the U.S. Y-STR Database, asearchable listing of 11- to 23-locus Y-STR haplotypes. The database isfunded by the National Institute of Justice and managed by the NationalCenter for Forensic Science (NCFS) in conjunction with the University ofCentral Florida. The U.S. Y-STR Database is a population database onlyand is intended for use in estimating Y-STR haplotype populationfrequencies for forensic case work purposes.

Several limitations exist for currently available Y-STR analysis kits.While haplotype databases are used to establish the frequency of ahaplotype in specific populations, haplotype resolution (HR) of kits mayvary across populations (Vermeulen et al., (2009) FSIG 3:205-213).Secondly, using current kits, a male relative of a suspected individualmay not be excluded. Relatives separated by up to 20 generations mayhave Y-STR profiles indistinguishable from each other, according tocurrent analyses (Ballantyne et al. (2010): Am J Hum Genet 87:341-353).Thirdly, adventitious matches increase as more male profiles are addedto Y-STR frequency databases. Therefore, there exists a need in the art,to improve Y-STR multiplex analysis systems, assays, kits, and methods.

SUMMARY OF SOME EMBODIMENTS OF THE INVENTION

In one aspect, the invention provides a set of amplification primersincluding primers for the amplification of at least 11 Y-STR markerswhere the primers are configured to provide each set of amplicons of theat least 11 Y-STR markers having a base pair size less than about 220base pairs. In some embodiments, detection of amplicon base pair sizemay be performed by a fluorescence detection technique. In someembodiments, detection of amplicon base pair size may be performed by amobility-dependent analytical technique. The mobility-dependentanalytical technique may be capillary electrophoresis. In some otherembodiments, detection of the amplicon base pair size may be performedby a sequencing technique using no fluorescent dye labels. In someembodiments, the set of amplification primers may further includeprimers for the amplification of at least 5 additional Y-STR markerswhere the primers are configured to provide each set of amplicons of theat least 5 additional Y-STR markers having a base pair size greater thanabout 220 base pairs. In various embodiments, when the set ofamplification primers amplify more than 11 Y-STR markers, then the setof amplification primers may be configured to provide all of the sets ofamplicons of the more than 11 Y-STR markers having a base pair size lessthan about 410 base pairs. In various embodiments, when the set ofamplification primers amplify more than 11 Y-STR markers, then the setof amplification primers may be configured to provide all of the sets ofamplicons of the more than 11 Y-STR markers having a base pair size lessthan about 420 base pairs. In some embodiments, when the set ofamplification primers amplify more than 11 Y-STR markers, then theamplification primer set may include primers for 25 Y-STR markers. Insome embodiments, when the amplification primer set includes primers for25 Y-STR markers, the set of amplification primers may include primersfor at least two double copy markers. In some embodiments, the set ofamplification primers may be labeled with one of at least 5 fluorescentdyes. In some embodiments, the set of amplification primers may beconfigured to provide each set of the amplicons of the at least 11 Y-STRmarkers labeled with one of at least 5 fluorescent dyes. The at least 5fluorescent dyes used to label the primers and/or the amplicons may beconfigured to be spectrally distinct. The set of amplification primersmay further include at least one amplification primer that includes amobility modifier. The set of amplification primers for theamplification of at least 11 Y-STR markers may be configured to provideat least one set of amplicons of the Y-STR markers including a mobilitymodifier. In some embodiments, the set of amplification primersamplifying at least 11 Y-STR markers, may amplify DYS576, DYS389I,DYS460, DYS458, DYS19, DYS456, DYS390, DYS570, DYS437, DYS393, andDYS439. In other embodiments, the set of amplification primersamplifying the at least 11 Y-STR markers configured to provide each setof amplicons of the at least 11 Y-STR markers having a base pair sizeless than about 220 base pairs, may amplify at least 5 Y-STR markerswhich are rapidly mutating loci. In some embodiments, the at least 5rapidly mutating Y-STR markers may include DYF387S1ab, DYS449, DYS570,DYS576, and DYS627. In other embodiments, the at least 5 rapidlymutating Y-STR markers may further include DYS518. In some embodiments,the set of primers for the amplification of at least 11 Y-STR markersmay be a set of primers for the amplification of DYF387S1ab, DYS19,DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS460,DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS481, DYS518,DYS533, DYS570, DYS576, DYS627, DYS635, and Y-GATA-H4. In otherembodiments, the set of primers for the amplification of at least 11Y-STR markers may be a set of primers for the amplification ofDYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392,DYS393, DYS460, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458,DYS481, DYS533, DYS570, DYS576, DYS627, DYS635, DYS643, and Y-GATA-H4.

In another aspect of the invention, a kit is provided for co-amplifyinga set of loci of at least one DNA sample including primers for theamplification of at least 11 Y-STR markers where the primers areconfigured to provide each set of amplicons of the at least 11 Y-STRmarkers having a base pair size less than about 220 base pairs; andoptionally, a size standard. In some embodiments, the kit may furtherinclude primers for the amplification of at least 5 Y-STR markers wherethe primers are configured to provide each set of amplicons of the atleast 5 Y-STR markers having a base pair size greater than about 220base pairs. The kit may include an amplification primer set for 25 Y-STRmarkers. In various embodiments, when the set of amplification primersamplify more than 11 Y-STR markers, then the set of amplificationprimers may be configured to provide all of the sets of amplicons of themore than 11 Y-STR markers having a base pair size less than about 410base pairs. In various embodiments, when the set of amplificationprimers amplify more than 11 Y-STR markers, then the set ofamplification primers may be configured to provide all of the sets ofamplicons of the more than 11 Y-STR markers having a base pair size lessthan about 420 base pairs. In some embodiments, the kit may include aset of amplification primers labeled with one of at least 5 fluorescentdyes. The at least 5 fluorescent dyes used to label the primers of thekit may be configured to be spectrally distinct. The kit may furtherinclude at least one amplification primer that includes a mobilitymodifier. In some embodiments, the kit including a set of amplificationprimers amplifying at least 11 Y-STR markers, may amplify DYS576,DYS389I, DYS460, DYS458, DYS19, DYS456, DYS390, DYS570, DYS437, DYS393,and DYS439. In other embodiments, the kit including a set ofamplification primers amplifying the at least 11 Y-STR markers, wherethe primers are configured to provide each set of amplicons of the atleast 11 Y-STR markers having a base pair size less than about 220 basepairs, may amplify at least 5 Y-STR markers which are rapidly mutatingloci. In some embodiments, the at least 5 rapidly mutating Y-STR markersmay include DYF387S1ab, DYS449, DYS570, DYS576, and DYS627. In otherembodiments, the at least 5 rapidly mutating Y-STR markers may furtherinclude DYS518. In some embodiments, the kit including a set of primersfor the amplification of at least 11 Y-STR markers may be a set ofprimers for the amplification of DYF387S1ab, DYS19, DYS385ab, DYS389I,DYS389II, DYS390, DYS391, DYS392, DYS393, DYS460, DYS437, DYS438,DYS439, DYS448, DYS449, DYS456, DYS458, DYS481, DYS518, DYS533, DYS570,DYS576, DYS627, DYS635, and Y-GATA-H4. In other embodiments, the kitincluding a set of primers for the amplification of at least 11 Y-STRmarkers may be a set of primers for the amplification of DYF387S1ab,DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393,DYS460, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS481,DYS533, DYS570, DYS576, DYS627, DYS635, DYS643, and Y-GATA-H4. In someembodiments, when the kit includes a size standard, the kit furtherincludes an allelic ladder.

In another aspect of the invention, a method is provided to amplifyalleles of Y-STR markers of a human male including the steps of:contacting a sample suspected to contain a DNA sample of a human malewith a set of amplification primers including primers for theamplification of the alleles of at least 11 Y-STR markers; andamplifying the sample thereby forming a plurality of sets of ampliconsof the at least 11 Y-STR markers where each set of the amplicons has abase pair size less than about 220 base pairs. The method may furtherinclude the step of detecting each set of amplicons whereby the allelesof the at least 11 Y-STR markers are identified. In some embodiments,the detecting step is performed by separating the plurality of sets ofamplicons using a mobility dependent analysis, where the plurality ofsets of amplicons is fluorescently labeled. In other embodiments, thedetecting step does not detect fluorescence. In embodiments, when thedetecting step does not detect fluorescence, the detecting step mayinclude ion semiconductor detection, pyrophosphate release detection, ormass spectrometry detection. In various embodiments of the method, theset of amplification primers may further include primers for theamplification of at least 5 additional Y-STR markers where the primersmay be configured to provide each set of amplicons of the at least 5additional Y-STR markers having a base pair size greater than about 220base pairs. In various embodiments of the method, when the set ofamplification primers amplifies more than 11 Y-STR markers, then the setof primers may be configured to provide all of the sets of amplicons ofthe more than 11 Y-STR markers having a base pair size less than about410 base pairs. In various embodiments of the method, when the set ofamplification primers amplifies more than 11 Y-STR markers, then the setof primers may be configured to provide all of the sets of amplicons ofthe more than 11 Y-STR markers having a base pair size less than about420 base pairs. In some embodiments of the method, the amplificationprimer set may include 25 Y-STR markers. In some embodiments, the set ofamplification primers may be labeled with one of at least 5 fluorescentdyes. In some other embodiments, each set of the amplicons of the atleast 11 Y-STR markers may be labeled with one of at least 5 fluorescentdyes. In various embodiments of the method, the at least 5 fluorescentdyes used to label the primers and/or the amplicons may be configured tobe spectrally distinct. The set of amplification primers used in themethod may further include at least one amplification primer thatincludes a mobility modifier. In some embodiments of the method, the atleast one set of amplicons may include a mobility modifier. In variousembodiments of the methods, the set of amplification primers amplifyingat least 11 Y-STR markers, may amplify DYS576, DYS389I, DYS460, DYS458,DYS19, DYS456, DYS390, DYS570, DYS437, DYS393, and DYS439. In otherembodiments, the set of amplification primers amplifying the at least 11Y-STR markers, may amplify at least 5 Y-STR markers which are rapidlymutating loci. In some embodiments, the at least 5 rapidly mutatingY-STR markers may include DYF387S1ab, DYS449, DYS570, DYS576, andDYS627. In other embodiments, the at least 5 rapidly mutating Y-STRmarkers may further include DYS518. In some embodiments of the method,the set of primers for the amplification of at least 11 Y-STR markersmay be a set of primers for the amplification of DYF387S1ab, DYS19,DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS460,DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS481, DYS518,DYS533, DYS570, DYS576, DYS627, DYS635, and Y-GATA-H4. In otherembodiments, the set of primers for the amplification of at least 11Y-STR markers may be a set of primers for the amplification ofDYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392,DYS393, DYS460, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458,DYS481, DYS533, DYS570, DYS576, DYS627, DYS635, DYS643, and Y-GATA-H4.In some embodiments, the method includes a set of amplification primersfor the amplification of the alleles of 27 Y-STR markers.

In yet another aspect, the invention provides a set of amplificationprimers including primers for the amplification of at least 22 Y-STRmarkers where at least 5 of the Y-STR markers are rapidly mutating loci.In some embodiments, the at least 5 rapidly mutating Y-STR markersinclude DYF387S1ab, DYS449, DYS570, DYS576, and DYS627. In someembodiments, the at least 5 rapidly mutating Y-STR markers includeDYS518. In various embodiments, the set of amplification primersconfigured to amplify the at least 22 Y-STR markers may be furtherconfigured to provide each set of amplicons of at least 11 Y-STR markershaving a base pair size less than about 220 base pairs. In otherembodiments, the set of amplification primers for the amplification ofat least 22 Y-STR markers may be configured to provide sets of ampliconsfor the at least 22 Y-STR markers each having a base pair size of lessthan about 410 base pairs. In other embodiments, the set ofamplification primers for the amplification of at least 22 Y-STR markersmay be configured to provide sets of amplicons for the at least 22 Y-STRmarkers each having a base pair size of less than about 420 base pairs.In some embodiments, detection of amplicon base pair size may beperformed by fluorescence detection. In some embodiments, detection ofamplicon base pair size may be performed by a mobility-dependentanalytical technique. The mobility-dependent analytical technique may becapillary electrophoresis. In some other embodiments, detection of theamplicon base pair size may be performed by a sequencing technique usingno detection of fluorescent dye labels. The amplification primer set mayinclude 25 Y-STR markers. In some embodiments, the set of amplificationprimers is labeled with one of at least 5 fluorescent dyes. In someembodiments, each set of the amplicons of the at least 22 Y-STR markersis labeled with one of at least 5 fluorescent dyes. The at least 5fluorescent dyes used to label the primers and/or the amplicons may beconfigured to be spectrally distinct. The set of amplification primersmay further include at least one amplification primer that includes amobility modifier. The set of amplification primers for theamplification of at least 22 Y-STR markers may be configured to provideat least one set of amplicons of the Y-STR markers where the at leastone set of amplicons includes a mobility modifier. In some embodiments,the at least 22 Y-STR markers may include DYF387S1ab, DYS19, DYS385ab,DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438,DYS439, DYS448, DYS449, DYS456, DYS458, DYS570, DYS576, DYS627, DYS635,and Y-GATA-H4. In other embodiments, the at least 22 Y-STR markers mayinclude DYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391,DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458,DYS518, DYS570, DYS576, DYS627, DYS635, and Y-GATA-H4. A kit forco-amplifying a set of loci of at least one DNA sample may be provided,including a set of amplification primers for the amplification of atleast 22 Y-STR markers where at least 5 of the Y-STR markers are rapidlymutating loci; and optionally, a size standard. In some embodiments, thesize standard is an allelic ladder.

In yet another aspect, a method is provided to amplify alleles of Y-STRmarkers of a human male including the steps of: contacting a samplewhich may contain a DNA sample of a human male with a set ofamplification primers including primers for the amplification of thealleles of at least 22 Y-STR markers, wherein at least 5 of the Y-STRmarkers are rapidly mutating loci; and amplifying the sample therebyforming a plurality of sets of amplicons of the at least 22Y-STRmarkers. In some embodiments of the method, a set of amplificationprimers of the alleles of at least 23 Y-STR markers are provided,wherein at least 5 of the Y-STR markers are rapidly mutating loci. Inyet other embodiments, a set of amplification primers of the alleles of27 Y-STR markers are provided, wherein at least 5 of the Y-STR markersare rapidly mutating loci. In some embodiments, the 27 Y-STR markersinclude 2 Y-STR markers having double copy markers contributing to thetotal number of Y-STR markers. In some embodiments, each set of theamplicons of at least 11 of the at least 22 Y-STR markers has a basepair size less than about 220 base pairs. In other embodiments, each setof the amplicons of at least 11 of at least 23 Y-STR markers has a basepair size less than about 220 base pairs. In yet other embodiments, eachset of the amplicons of at least 11 of 27 Y-STR markers has a base pairsize less than about 220 base pairs. In various embodiments of themethod, a set of amplification primers including primers for theamplification of the alleles of at least 22 Y-STR markers are provided,wherein at least 6 of the Y-STR markers are rapidly mutating loci. Invarious embodiments of the method, a set of amplification primersincluding primers for the amplification of the alleles of at least 22Y-STR markers are provided, wherein at least 7 of the Y-STR markers arerapidly mutating loci. The method may further include the step ofdetecting each set of amplicons whereby the alleles of at least 22 Y-STRmarkers are identified. In some embodiments, the alleles of at least 23Y-STR markers are identified. In yet other embodiments, the alleles of27 Y-STR markers are identified. In some embodiments, the detecting stepis a fluorescence detection step. In some embodiments, the detectingstep is performed by separating the plurality of sets of amplicons usinga mobility dependent analysis, where the plurality of sets of ampliconsis fluorescently labeled. In other embodiments, the detecting step doesnot detect fluorescence. In embodiments, when detecting steps do notdetect fluorescence, the detecting step may include ion semiconductordetection, pyrophosphate release detection, or mass spectrometrydetection. In some embodiments, the at least 11 Y-STR markers havingamplicons having a base pair size of less than about 220 base pairs areDYS576, DYS389I, DYS460, DYS458, DYS19, DYS456, DYS390, DYS570, DYS437,DYS393, and DYS439. In some the embodiments, the at least 5 rapidlymutating Y-STR markers are selected from the group consisting ofDYF387S1ab, DYS449, DYS518, DYS570, DYS576, and DYS627. In otherembodiments, the at least 5 rapidly mutating Y-STR markers are 6 rapidlymutating Y-STR markers.

In another aspect, a method of male individual identification isprovided, including the steps of: contacting a sample containing anucleic acid of a human male with a set of amplification primersincluding primers for the amplification of the alleles of at least 11Y-STR markers; and amplifying the sample thereby forming a plurality ofsets of amplicons of the at least 11 Y-STR markers where each set of theamplicons has a base pair size less than about 220 base pairs; anddetecting each set of amplicons whereby the alleles of the maleindividual are identified. In various embodiments of the methods, theset of amplification primers amplifying at least 11 Y-STR markers, mayamplify DYS576, DYS389I, DYS460, DYS458, DYS19, DYS456, DYS390, DYS570,DYS437, DYS393, and DYS439. In other embodiments, the step of amplifyingthe at least 11 Y-STR markers, may include amplifying at least 5 Y-STRmarkers which are rapidly mutating loci. In some embodiments, the atleast 5 rapidly mutating Y-STR markers may include DYF387S1ab, DYS449,DYS570, DYS576, and DYS627. In other embodiments, the at least 5 rapidlymutating Y-STR markers may further include DYS518. In some embodimentsof the method, the set of primers for the amplification of at least 11Y-STR markers may be a set of primers for the amplification ofDYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392,DYS393, DYS460, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458,DYS481, DYS518, DYS533, DYS570, DYS576, DYS627, DYS635, and Y-GATA-H4.In other embodiments, the set of primers for the amplification of atleast 11 Y-STR markers may be a set of primers for the amplification ofDYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392,DYS393, DYS460, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458,DYS481, DYS533, DYS570, DYS576, DYS627, DYS635, DYS643, and Y-GATA-H4.In some embodiments, the method includes a set of amplification primersfor the amplification of the alleles of more than 11Y-STR markers. Inother embodiments, the plurality of sets of amplicons of the more than11 Y-STR markers where the plurality of sets of the amplicons has a basepair size less than about 410 base pairs. In some embodiments, thedetecting step is a fluorescence detection step. In some embodiments,the method further includes the step of comparing the alleles identifiedfor a first male individual to the alleles identified for a second maleindividual, whereby the first male individual is differentiable from thesecond male individual. In some embodiments, the first male individualhas a similar paternal genetic lineage as the second male individual.

These embodiments and other features of the present teachings willbecome more apparent from the description herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of selected Y-STR markers, whereGene Diversity values are graphed on the x axis and mutation rate ismapped along the y axis.

FIG. 2 is a schematic representation of one embodiment of a Y-STRmultiplex assay panel, Panel 1.

FIG. 3 is a schematic representation of an embodiment of a Y-STRmultiplex assay panel, Panel 2.

FIG. 4 is a schematic representation of another embodiment of a Y-STRmultiplex assay panel, Panel 3.

FIG. 5 is a schematic representation of a further embodiment of a Y-STRmultiplex assay panel, Panel 4.

FIG. 6 is a schematic representation of yet another embodiment of aY-STR multiplex assay panel, Panel 5.

FIG. 7 is a schematic representation of yet another embodiment of aY-STR multiplex assay panel, Panel 6.

FIG. 8 is a graphical representation of an electrophoretic run for theY-STR panel of FIG. 7 (Panel 6), color separated. The 6^(th) dyestandard channel is not shown.

FIG. 9 is a table comparing selected Y-STR marker panels from varioussources to Panel 6 and includes the number of alleles for each panel.

FIG. 10 is a schematic representation of another embodiment of a Y-STRmultiplex assay panel, Panel 7.

FIG. 11 is a graphical representation of an electrophoretic run for theY-STR panel of FIG. 10 (Panel 7), color separated. The 6^(th) dyestandard channel is not shown

FIG. 12 is a table comparing selected Y-STR marker panels from varioussources to Panel 7 and includes the number of alleles for each panel.

FIG. 13 is a graphical representation comparing the alleles of theYfiler® multiplex assay and the expanded alleles of Panels 1-7 forDYS438 marker.

FIG. 14 is a graphical representation of the effect of primer length forDYS456 marker when amplifying selected male/female DNA mixtures.

FIG. 15A is a graphical representation of one embodiment of an allelicladder for the multiplex panel of FIG. 10 (Panel 7).

FIG. 15B is a graphical representation of another embodiment of anallelic ladder for the multiplex panel of FIG. 10 (Panel 7).

FIG. 16 is a graphical representation comparing the number of allelesrecovered from PCR amplifications using Y-STR Panel 6 and Yfiler®, asdecreasing amounts of target DNA are used.

FIG. 17 is a graphical representation comparing the number of allelesrecovered from PCR amplifications using Y-STR Panel 7 and Yfiler®, asdecreasing amounts of target DNA are used.

FIG. 18 is a graphical representation comparing the percentage ofalleles identified using the Y-STR Panel 6 and Yfiler® multiplexes asthe ratio of male to female DNA decreases, using a constantconcentration of female DNA.

FIG. 19 is a graphical representation comparing the percentage ofalleles identified using the Y-STR Panel 7 and Yfiler® multiplexes asthe ratio of male to female DNA decreases, using a constantconcentration of female DNA.

FIG. 20 is a graphical representation comparing the percentage ofalleles identified using the Y-STR Panel 6 and Yfiler® multiplexes asthe ratio of male to female DNA decreases, using an increasingconcentration of female DNA.

FIG. 21 is a graphical representation of the comparison of theintracolor balance when using Panel 6 multiplex or the Yfiler®multiplex.

FIG. 22 is a graphical representation of the comparison of theintracolor balance when using Panel 7 multiplex or the Yfiler®multiplex, at two different ratios of male:female DNA.

FIG. 23 is a graphical representation of the comparison of the averagepercentage of alleles identified using the Y-STR Panel 6 or Yfiler®multiplexes when amplifying target DNA in the presence of increasingamounts of humic acid.

FIG. 24 is a graphical representation of the comparison of the averagepercentage of alleles identified using the Y-STR Panel 6 or Yfiler®multiplexes when amplifying target DNA in the presence of increasingamounts of Hematin.

FIG. 25 is a graphical representation of the comparison of the averagepercentage of alleles identified using the Y-STR Panel 7 or Yfiler®multiplexes when amplifying target DNA in the presence of increasingamounts of Hematin or Humic acid.

FIG. 26 is a graphical representation of the comparison of theintracolor balance for Panel 7 or Yfiler® multiplexes when analysis isperformed in the presence of two different concentrations of hematin orhumic acid.

FIG. 27 is a graphical representation of an electropherogram showing theresolution of a Male/Male mixture using the Y-STR Panel 6 multiplex.

FIG. 28 is a graphical representation of the intracolor balance ofelectrophoretic signals provided by direct amplification of biologicalsamples on selected substrates using the Panel 7 multiplex.

FIG. 29 is a graphical representation of an electropherogram of theamplification results of one of the directly amplified samples of FIG.28.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

For the purposes interpreting of this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with the usage of that word inany other document, including any document incorporated herein byreference, the definition set forth below shall always control forpurposes of interpreting this specification and its associated claimsunless a contrary meaning is clearly intended (for example in thedocument where the term is originally used). It is noted that, as usedin this specification and the appended claims, the singular forms “a,”“an,” and “the,” include plural referents unless expressly andunequivocally limited to one referent. The use of “or” means “and/or”unless stated otherwise. For illustration purposes, but not as alimitation, “X and/or Y” can mean “X” or “Y” or “X and Y”. The use of“comprise,” “comprises,” “comprising,” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.Furthermore, where the description of one or more embodiments uses theterm “comprising,” those skilled in the art would understand that, insome specific instances, the embodiment or embodiments can bealternatively described using the language “consisting essentially of”and/or “consisting of”. The term “and/or” means one or all of the listedelements or a combination of any two or more of the listed element.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way. All literature cited in this specification, including but notlimited to, patents, patent applications, articles, books, and treatisesare expressly incorporated by reference in their entirety for anypurpose. In the event that any of the incorporated literaturecontradicts any term defined herein, this specification controls. Whilethe present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

Any material, or portion thereof, that is said to be incorporated byreference herein, but which conflicts with existing definitions,statements, or other disclosure material explicitly set forth herein isonly incorporated to the extent that no conflict arises between thatincorporated material and the present disclosure material. In the eventof a conflict, the conflict is to be resolved in favor of the presentdisclosure as the preferred disclosure.

The practice of the present invention may employ conventional techniquesand descriptions of organic chemistry, polymer technology, molecularbiology (including recombinant techniques), cell biology, biochemistry,and immunology, which are within the skill of the art. Such conventionaltechniques include oligonucleotide synthesis, hybridization, extensionreaction, and detection of hybridization using a label. Specificillustrations of suitable techniques can be had by reference to theexample herein below. However, other equivalent conventional procedurescan, of course, also be used. Such conventional techniques anddescriptions can be found in standard laboratory manuals such as GenomeAnalysis: A Laboratory Manual Series (Vols. I-IV), PCR Primer: ALaboratory Manual, and Molecular Cloning: A Laboratory Manual (all fromCold Spring Harbor Laboratory Press, 1989), Gait, “OligonucleotideSynthesis: A Practical Approach” 1984, IRL Press, London, Nelson and Cox(2000), Lehninger, Principles of Biochemistry 3^(rd) Ed., W. H. FreemanPub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y. all of which are herein incorporated intheir entirety by reference for all purposes.

The term “allele” as used herein refers to a genetic variationassociated with a gene or a segment of DNA, i.e., one of two or morealternate forms of a DNA sequence occupying the same locus. In someembodiments, an allele within a locus encompasses a nucleic acidmolecule having a polymorphic tandemly repeated base pair motif. It isthe variation in the number of repeat units in tandem that distinguishalleles within a locus.

The term “wild type allele” or “predominant allele” are usedinterchangeably herein and as used herein refer to the most frequentlyoccurring allele found in a given species, genus, family, segment,tribe, ethnicity, or racial population. The wild type allele can beconsidered the most common allele.

The term “variant allele” as used herein refers to a variation from themost frequently occurring allele. It can also refer to, at one or morenucleic acid positions, a change in the nucleic acid sequence at one ormore positions resulting in one or more differences when compared to themost common allele at one or more nucleic acid positions as found in theallele for a given species, genus, family, segment, tribe, ethnicity, orracial population.

The term “allelic ladder” as used herein refers to a nucleic acid sizestandard that encompasses size standards for one or more alleles for aparticular STR marker. The allelic ladder serves as a reference standardand nucleic acid size marker for the amplified allele(s) from the STRmarker.

As used herein, the terms “amplification primer” and “oligonucleotideprimer” are used interchangeably and refer to an oligonucleotide,capable of annealing to an RNA or DNA region adjacent a target sequence,and serving as an initiation primer for DNA synthesis under suitableconditions well known in the art. Typically, a PCR reaction employs an“amplification primer pair” also referred to as an “oligonucleotideprimer pair” including an “upstream” or “forward” primer and a“downstream” or “reverse” primer, which delimit a region of the RNA orDNA to be amplified. A first primer and a second primer may be either aforward or reverse primer and are used interchangeably herein and arenot to be limiting.

As used herein, “amplify” refers to the process of enzymaticallyincreasing the amount of a specific nucleotide sequence. Thisamplification is not limited to but is generally accomplished by PCR. Asused herein, “denaturation” refers to the separation of twocomplementary nucleotide strands from an annealed state. Denaturationcan be induced by a number of factors, such as, for example, ionicstrength of the buffer, temperature, or chemicals that disrupt basepairing interactions. As used herein, “annealing” refers to the specificinteraction between strands of nucleotides wherein the strands bind toone another substantially based on complementarity between the strandsas determined by Watson-Crick base pairing. It is not necessary thatcomplementarity be 100% for annealing to occur. As used herein,“extension” refers to the amplification cycle after the primeroligonucleotide and target nucleic acid have annealed to one another,wherein the polymerase enzyme catalyzes primer extension, therebyenabling amplification, using the target nucleic acid as a replicationtemplate.

The terms “amplicon,” “amplification product” and “amplified sequence”are used interchangeably herein and refer to a broad range of techniquesfor increasing polynucleotide sequences, either linearly orexponentially and can be the product of an amplification reaction. Anamplicon can be double-stranded or single-stranded, and can include theseparated component strands obtained by denaturing a double-strandedamplification product. In certain embodiments, the amplicon of oneamplification cycle can serve as a template in a subsequentamplification cycle. Exemplary amplification techniques include, but arenot limited to, PCR or any other method employing a primer extensionstep. Other nonlimiting examples of amplification include, but are notlimited to, ligase detection reaction (LDR) and ligase chain reaction(LCR). Amplification methods can include thermal-cycling or can beperformed isothermally. In various embodiments, the term “amplificationproduct” and “amplified sequence” includes products from any number ofcycles of amplification reactions.

As used herein, the term “base pair motif” refers to the nucleobasesequence configuration including, but not limited to, a repetitivesequence, a sequence with a biological significance, a tandem repeatsequence, and so on.

As used herein, the term “comparing” broadly refers to differencesbetween two or more nucleic acid sequences. The similarity ordifferences can be determined by a variety of methods, including but notlimited to: nucleic acid sequencing, alignment of sequencing reads, gelelectrophoresis, restriction enzyme digests, single strandconformational polymorphism, and so on.

The terms “detecting” and “detection” are used in a broad sense hereinand encompass any technique by which one can determine the presence ofor identify a nucleic acid sequence. In some embodiments, detecting mayinclude quantitating a detectable signal from the nucleic acid,including without limitation, a real-time detection method, such asquantitative PCR (“Q-PCR”). In some embodiments, detecting may includedetermining the sequence of a sequencing product or a family ofsequencing products generated using an amplification product as thetemplate; in some embodiments, such detecting may include obtaining thesequence of a family of sequencing products. In other embodimentsdetecting can be achieved through measuring the size of a nucleic acidamplification product.

As used herein, “DNA” refers to deoxyribonucleic acid in its variousforms as understood in the art, such as genomic DNA, cDNA, isolatednucleic acid molecules, vector DNA, and chromosomal DNA. “Nucleic acid”refers to DNA or RNA in any form. Examples of isolated nucleic acidmolecules include, but are not limited to, recombinant DNA moleculescontained in a vector, recombinant DNA molecules maintained in aheterologous host cell, partially or substantially purified nucleic acidmolecules, and synthetic DNA molecules. Typically, an “isolated” nucleicacid is free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,is generally substantially free of other cellular material or culturemedium when produced by recombinant techniques, or free of chemicalprecursors or other chemicals when chemically synthesized.

As used herein, the term “flanking sequence” broadly refers to nucleicacid sequence 5′ and/or 3′ of a target nucleic acid sequence, including,but not limited to, a short tandem repeat sequence. The flankingsequence can be within an amplification product or outside, i.e.,flanking, the amplification product. Amplification primers can beselected to hybridize to sequences flanking the variable portion of anSTR marker so as to produce amplicons of a size indicative of a specificallele of the STR marker.

As used herein, the term “short tandem repeat (STR) loci” refers toregions of a genome which contains short, repetitive sequence elementsof 2 to 7 base pairs in length. Each sequence element is repeated atleast once within an STR and is referred to herein as a “repeat unit.”The term STR also encompasses a region of genomic DNA wherein more thana single repeat unit is repeated in tandem or with intervening bases,provided that at least one of the sequences is repeated at least twotimes in tandem. Examples of STRs, include but are not limited to, atriplet repeat, e.g., ATC in tandem, e.g., ATCATCATCATCAACATCATC (SEQ IDNO: 1); a 4-peat (tetra-repeat), e.g., GATA in tandem, e.g.,GATAGATAGATACATAGATA (SEQ ID NO: 2); and a 5-peat (penta-repeat), e.g.,ATTGC in tandem, e.g., ATTGCATTGCATTGC (SEQ ID NO: 3) and so on.Information about specific STRs that can be used as genetic markers canbe found in, among other places, the STRbase.

As used herein, the terms “imperfect repeat”, “incomplete repeat”, and“variant repeat” refer to a tandem repeat within which the repeat unit,though in tandem, has sequence interruptions (additions or deletions)between one or more repeat units, e.g., ATCG ATCG AACG ATCG ATCG (SEQ IDNO:4), where the third repeat unit is not identical to the other repeatunits and so an imperfect repeat; an incomplete repeat can be seen as atandem repeat in which the number of base pairs in a repeat unit is anincomplete repeat, e.g., allele 9 of the TH01 locus contains nine 4-peatrepeat units ([AATG]₉ for the complete repeat “AATG” for the TH01locus), but the 9.3 allele contains the nine “AATG” repeats and oneincomplete repeat, “ATG” of three nucleotides, an incomplete repeat,i.e., [AATG]₆ATG[AATG]₃; while a variant repeat has variation(s) withinthe repeat unit, e.g., ATCC ATCG ATCC ATCG ATCG ATCC ATCC (SEQ ID NO:5),where the 4-peat repeat unit has a variant base pair at the fourthposition of the repeat unit, either a “C” or a “G” nucleotide.

As used herein, the term “polymorphic short tandem repeat loci” refersto STR loci in which the number of repetitive sequence elements (and netlength of the sequence) in a particular region of genomic DNA variesfrom allele to allele, and from individual to individual.

“Genetic markers” are generally alleles of genomic DNA loci withcharacteristics of interest for analysis, such as DNA typing, in whichindividuals are differentiated based on variations in their DNA. MostDNA typing methods are designed to detect and analyze differences in thelength and/or sequence of one or more regions of DNA markers known toappear in at least two different forms, or alleles, in a population.Such variation is referred to as “polymorphism,” and any region of DNAin which such a variation occurs is referred to as a “polymorphiclocus.” One possible method of performing DNA typing involves thejoining of PCR amplification technology (K B Mullis, U.S. Pat. No.4,683,202) with the analysis of length variation polymorphisms. PCRtraditionally could only be used to amplify relatively small DNAsegments reliably; i.e., only amplifying DNA segments under 3,000 basesin length (M. Ponce and L. Micol (1992), NAR 20(3):623; R. Decorte etal. (1990), DNA CELL BIOL. 9(6):461 469). Short tandem repeats (STRs),minisatellites and variable number of tandem repeats (VNTRs) are someexamples of length variation polymorphisms. DNA segments containingminisatellites or VNTRs are generally too long to be amplified reliablyby PCR. By contrast STRs, containing repeat units of approximately threeto seven nucleotides, are short enough to be useful as genetic markersin PCR applications, because amplification protocols can be designed toproduce smaller products than are possible from the other variablelength regions of DNA.

As used herein, the term “haplotype” is a selected group of alleles on aY-chromosome that are transmitted together.

The term “locus” as used herein refers to a specific physical positionon a chromosome or a nucleic acid molecule. Alleles of a locus arelocated at identical sites on homologous chromosomes. “Loci” the pluralof “locus” as used herein refers to a specific physical position oneither the same or a different chromosome as well as either the same ora different specific physical position on the nucleic acid molecule.

As used herein, the term “nucleic acid sample” refers to nucleic acidfound in biological samples according to the present inventionincluding, but not limited to, for example, hair, feces, blood, tissue,urine, saliva, cheek cells, vaginal cells, skin, for example skin cellscontained in fingerprints, bone, tooth, buccal sample, amniotic fluidcontaining placental cells, and amniotic fluid containing fetal cellsand semen. It is contemplated that samples may be collected invasivelyor noninvasively. The sample can be on, in, within, from or found inconjunction with a fiber, fabric, cigarette, chewing gum, adhesivematerial, soil or inanimate objects. “Sample” as used herein, is used inits broadest sense and refers to a sample suspected of containing anucleic acid and may entail a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA, RNA, cDNA andthe like. Samples can be of animal or vegetable origins encompassing anyorganism containing nucleic acid, including, but not limited to,bacteria, viruses, plants, livestock, household pets, and human samples.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

As used herein, the “polymerase chain reaction” or PCR is a anamplification of nucleic acid consisting of an initial denaturation stepwhich separates the strands of a double stranded nucleic acid sample,followed by repetition of (i) an annealing step, which allowsamplification primers to anneal specifically to positions flanking atarget sequence; (ii) an extension step which extends the primers in a5′ to 3′ direction thereby forming an amplicon polynucleotidecomplementary to the target sequence, and (iii) a denaturation stepwhich causes the separation of the amplicon from the target sequence(Mullis et al., eds, The Polymerase Chain Reaction, BirkHauser, Boston,Mass. (1994)). Each of the above steps may be conducted at a differenttemperature, preferably using an automated thermocycler (AppliedBiosystems LLC, a division of Life Technologies Corporation, FosterCity, Calif.). If desired, RNA samples can be converted to DNA/RNAheteroduplexes or to duplex cDNA by methods known to one of skill in theart. The PCR method also includes reverse transcriptase-PCR and otherreactions that follow principles of PCR.

As used herein, the terms “polynucleotide”, “oligonucleotide”, and“nucleic acid” are used interchangeably herein and refer tosingle-stranded and double-stranded polymers of nucleotide monomers,including without limitation 2′-deoxyribonucleotides (DNA) andribonucleotides (RNA) linked by internucleotide phosphodiester bondlinkages, or internucleotide analogs, and associated counter ions, e.g.,H⁺, NH₄ ⁺, trialkylammonium, Mg²⁺, Na⁺, and the like. A polynucleotidemay be composed entirely of deoxyribonucleotides, entirely ofribonucleotides, or chimeric mixtures thereof and can include nucleotideanalogs. The nucleotide monomer units may include any nucleotide ornucleotide analog. Polynucleotides typically range in size from a fewmonomeric units, e.g. 5-40 when they are sometimes referred to in theart as oligonucleotides, to several thousands of monomeric nucleotideunits. Unless denoted otherwise, whenever a polynucleotide sequence isrepresented, it will be understood that the nucleotides are in 5′ to 3′order from left to right and that “A” denotes deoxyadenosine, “C”denotes deoxycytosine, “G” denotes deoxyguanosine, “T” denotesthymidine, and “U” denotes deoxyuridine, unless otherwise noted.

The term “primer” refers to a polynucleotide (oligonucleotide) andanalogs thereof that is capable of selectively hybridizing to a targetnucleic acid or “template,” a target region flanking sequence or to acorresponding primer-binding site of an amplification product; andallows the synthesis of a sequence complementary to the correspondingpolynucleotide template, flanking sequence or amplification product fromthe primer's 3′ end. Typically a primer can be between about 10 to 100nucleotides in length and can provide a point of initiation fortemplate-directed synthesis of a polynucleotide complementary to thetemplate, which can take place in the presence of appropriate enzyme(s),cofactors, substrates such as nucleotides (dNTPs) and the like.

As used herein, the term “primer-binding site” refers to a region of apolynucleotide sequence, typically a sequence flanking a target regionand/or an amplicon that can serve directly, or by virtue of itscomplement, as the template upon which a primer can anneal for anysuitable primer extension reaction known in the art, for example, butnot limited to, PCR. It will be appreciated by those of skill in the artthat when two primer-binding sites are present on a double-strandedpolynucleotide, the orientation of the two primer-binding sites isgenerally different. For example, one primer of a primer pair iscomplementary to and can hybridize with the first primer-binding site,while the corresponding primer of the primer pair is designed tohybridize with the complement of the second primer-binding site. Statedanother way, in some embodiments the first primer-binding site can be ina sense orientation, and the second primer-binding site can be in anantisense orientation. A primer-binding site of an amplicon may, butneed not, encompass the same sequence as or at least some of thesequence of the target flanking sequence or its complement.

Those in the art understand that as a target region is amplified bycertain amplification means, the complement of the primer-binding siteis synthesized in the complementary amplicon or the complementary strandof the amplicon. Thus, it is to be understood that the complement of aprimer-binding site is expressly included within the intended meaning ofthe term primer-binding site, as used herein.

As used herein, the term “tandem repeat” refers to a repetitive sequenceoccurring in sequential succession.

As used herein, the term “tandem repeat locus” refers to a locuscontaining tandem repeats.

As used herein, the terms “target polynucleotide,” “nucleic acid target”and “target nucleic acid” are used interchangeably herein and refer to aparticular nucleic acid sequence of interest. The “target” can be apolynucleotide sequence that is sought to be amplified and can exist inthe presence of other nucleic acid molecules or within a larger nucleicacid molecule. The target polynucleotide can be obtained from anysource, and can include any number of different compositionalcomponents. For example, the target can be nucleic acid (e.g. DNA orRNA). The target can be methylated, non-methylated, or both. Further, itwill be appreciated that “target polynucleotide” can refer to the targetpolynucleotide itself, as well as surrogates thereof, for exampleamplification products, and native sequences. In some embodiments, thetarget polynucleotide is a short DNA molecule derived from a degradedsource, such as can be found in, for example, but not limited to,forensics samples (see for example Butler, 2001, Forensic DNA Typing:Biology and Technology Behind STR Markers). The target polynucleotidesof the present teachings can be derived from any of a number of sources.These sources may include, but are not limited to, whole blood, a tissuebiopsy, lymph, bone, bone marrow, tooth, amniotic fluid, hair, skin,semen, anal secretions, vaginal secretions, perspiration, saliva, buccalswabs, various environmental samples (for example, agricultural, water,and soil), research samples generally, purified samples generally, andlysed cells. It will be appreciated that target polynucleotides can beisolated from samples using any of a variety of procedures known in theart, for example the PrepSEQ™ Kits (from Applied Biosystems), Boom etal., and U.S. Pat. No. 5,234,809, etc. It will be appreciated thattarget polynucleotides can be cut or sheared prior to analysis,including the use of such procedures as mechanical force, sonication,restriction endonuclease cleavage, or any method known in the art.

The nomenclature for the particular STR loci as used herein refer to thenames assigned to these loci as they are known in the art. The loci areidentified, for example, in the various references and by the variousaccession numbers in the list that follows, all of which areincorporated herein by reference in their entirety. The list ofreferences that follows is merely intended to be exemplary of sources oflocus information. Where appropriate, the current Accession Number as oftime of filing is presented, as provided by GenBank® (National Centerfor Biotechnology Information, Bethesda, Md.).

New Y-STR multiplex analysis panels are described here, which providesurprising improvements in the ability to provide haplotype resolution(HR) variation across more diverse populations, ability to exclude amale relative of a suspected individual, and/or ability to resolveadventitious matches in more highly populated Y-STR frequency databases.There is a need in the field for such improvements, as currently, theAmpFlSTR® Yfiler® multiplex analysis has a HR in European populations of0.989, but only a HR of 0.905 globally (Vermeulen, Forensic ScienceInternational Genetics 3 (2009) 205-213). Additionally, the Y-STRmultiplex panels described here provide better overall balance of themultiplex analysis identifying more robustly minor contributor allelesin a mixture, thus providing either better male/male resolution and/orbetter identification of male alleles in male/female mixtures with highfemale background, compared to commercially available kits. Further, theY-STR multiplex panels provide: improved resistance to inhibitors of PCRproviding higher recovery of alleles; higher sensitivity, providinghigher number of alleles identified when amplifying small amounts ofinput DNA; and shorter analysis times compared to the currentlyavailable commercial kits.

In the Y-STR multiplex assay panels described here, the Y-STR markerscurrently used in the Yfiler® multiplex panel have also been includedbecause existing Y-STR databases are already populated with profilescontaining this information. The use of additional Y-STR markers hasbeen evaluated. A large number of Y-STR loci have been identified butnot all Y-STR loci are necessarily suitable for inclusion in a multiplexpanel for a number of reasons. Multicopy markers may be challenging wheninterpreting data, especially in samples having several DNA sources orpotential contaminants. For example, markers with more than two copies(non-limiting examples include DYF399S1abs and DYF403S1 1abc/II) may notbe considered for inclusion in some embodiments of the invention.

Use of loci having higher gene diversity has been investigated todecrease the incidence of adventitious matches. For exclusion of closepatrilineal relatives of an individual, markers with a high mutationrate may be beneficial. On the other hand, in kinship analysis markerswith high mutation rates may complicate analysis; including additionalmarkers with lower mutation rates may aid in lineage differentiation.Balancing those factors may provide a multiplex panel with the broadestapplicability.

Newly added Y-STR markers provide improvements beyond the capability ofcurrently commercialized Y-STR multiplex assays, including features suchas (1) the use of mini-STRs which can facilitate analysis of degradedDNA, (2) the inclusion of highly discriminating markers which may betterdifferentiate paternal lineages in populations with low Y-chromosomediversity and (3) the use of rapidly mutating markers to increase theability to distinguish between close relatives. Additionally selectingmarkers having a maximum of two copies per marker (e.g. DYS385ab) maysimplify analysis.

Other factors contributing to selection of Y-STR loci for improvedmultiplex analysis include primer compatibility within the multiplex,strict male specificity for the associated primers, and potential foruse as a mini-STR. Another advantage of including mini-STRs is thepotential for shortening the time for the electrophoretic separationsused to identify the alleles that are amplified in the multiplex, asgreater numbers of STR markers may be included in an electrophoreticseparation of about 410 bp. Adding a sixth dye channel also permits anincrease in the number of Y-STR loci examined while maintaining ashorter electrophoretic separation.

Gene Diversity.

Average gene diversity (GD) values were gathered from availablepopulation studies as listed in TABLE 1 for a candidate list of 39 loci.These 39 loci were further evaluated for the possibility of inclusion inthe improved Y-STR multiplex panels, as these loci offer the potentialfor greater discrimination, particularly in non-European populations, asshown in the key to TABLE 1.

TABLE 1 Ave. Mutation No. Marker GD Gene Diversity (GD) Rate(3) 1DYF387S1 ab 0.950 0.950[W3] 0.0159 2 DYF404S1 ab 0.920 0.920[w3] 0.01253 DYF406S1 0.741 0.741[EU1] 0.0038 4 DYS19 0.655 0.747[W1], 0.676[US2],0.457[Cau3], 0.718[AA3], 0.0044 0.688[CN1], 0.700[KR1], 0.683[KR2],0.717[KR3], 0.688[JP1], 0.758[PL1], 0.700[BY1], 0.516[ES1], 0.563[ES4],0.688[EU1], 0.535[NL], 0.638[CZ1], 0.694[CA1], 0.691[MZ1], 0.480[ZA-E1],0.700[ZA-I1], 0.72[ZA-X1] 5 DYS385 ab 0.887 0.973[W1], 0.912[US2],0.541[Cau3], 0.553[AA3], 0.0031 0.793[CN1], 0.963[KR1], 0.961[KR2],0.948[JP1], 0.875[PL1], 0.845[BY1], 0.800[ES1], 0.840[ES4], 0.875[EU1],0.843[NL1], 0.820[CZ1], 0.933[CA1], 0.922[MZ1] 6 DYS388 0.435 0.360[W1],0.365[US2], 0.330[US3], 0.437[CN1], 0.0004 0.536[KR1], 0.509[KR2],0.5083[KR3], 0.400[PL1], 0.449[BY1], 0.334[ES1], 0.553[NL1] 7 DYS389 I0.581 0.669[W1], 0.549[US2], 0.541[Cau3], 0.553[AA3], 0.0055 0.582[CN1],, 0.718[KR1], 0.667[KR2], 0.666[KR3], 0.646[JP1], 0.460[BY1],0.563[ES1], 0.575[ES4], 0.5498[EU1], 0.544[NL1], 0.546[CZ1], 0.555[CA1],0.495[MZ1] 8 DYS389 II 0.691 0.724[W1], 0.736[US2], 0.590[Cau3],0.701[AA3], 0.0038 0.767[CN1], 0.690[KR1], 0.735[KR2], 0.726[KR3],0.769[JP1], 0.799[PL1], 0.676[BY1], 0.507[ES1], 0.563[ES4], 0.764[[EU1],0.53[NL1], 0.780[CZ1], 0.669[CA1], 0.714[MZ1] 9 DYS390 0.672 0.789[W1],0.764[US2], 0.704[Cau3], 0.659[AA3], 0.0015 0.696[CN1], 0.673[KR1],0.626[KR2], 0.669[KR3], 0.765[JP1], 0.654[PL1], 0.687[ES1], 0.573[ES2],0.606[ES4], 0.752[EU1], 0.681[NL1], 0.691[CZ1], 0.497[CA1], 0.528[MZ1],0.680[ZA-E1], 0.830[ZA-I1], 0.580[ZA-X1] 10 DYS391 0.444 0.532[W1],0.534[US2], 0.522[Cau3], 0.417[AA3], 0.0032 0.393[CN1], 0.616[KR1],0.403[KR2], 0.292[KR3], 0.209[JP1], 0.504[PL1], 0517[BY1], 0594[ES1],0561[ES4, 0.509[EU1], 0.527[NL1], 0.541[CZ1], 0.399[CA1], 0.305[MZ1],0.540[ZA-E1], 0.280[ZA-I1], 0.12[ZA-X1] 11 DYS392 0.509 0.768[W1],0.609[US2], 0.602[Cau3], 0.416[AA3], 0.0010 0.642[CN1], 0.691[KR1],0.684[KR2], 0.693[KR3], 0.651[JP1], 0.321[PL1], 0.346[BY1], 0.501[ES1],0.565[ES4], 0.5782[EU1], 0.618[CZ1], 0.391[CA1], 0.018[MZ1], 0.580[ZA-E1], 0.450[ZA-I1], 0.050 [ZA- X1] 12 DYS393 0.522 0.664[W1],0.485[US2], 0.322[Cau3], 0.608[AA3], 0.0021 0.616[CN1], 0.659[KR1],0.638[KR2], 0.634[ KR3], 0.556[JP1], 0.342[PL1], 0.381[ES1], 0.463[ES4],0.457[EU1], 0.410[NL1], 0.503[CZ1], 0.670[CA1], 0.310[ZA-E1],0.690[ZA-I1], 0.580[ZA-X1] 13 DYS437 0.477 0.565[W1], 0.637[US2],0.610[US3], 0.415[KR1], 0.0015 0.255[JP1], 0.457[PL1], 0.464[BY1],0.543[ES1], 0.575[ES2], 0.602[ES3], 0.574[ES4], 0.644[EU1], 0.581[NL1],0.584[BR1], 0.312[CA1], 0.053[MZ1], 0.546[ZA-I2], 0.551[ZA-E2],0.090[ZA-X2] 14 DYS438 0.583 0.598[W1], 0.691[US2], 0.617[Cau3],0.498[AA3] 0.0010 0.467[CN3], 0.527[CN5], 0.640[KR1], 0.607[JP1],0.584[PL1], 0.578[BY1], 0.540[ES2], 0.554[ES3], 0.589[ES4], 0.694[EU1],0.584[NL1], 0.703[CZ1], 0.690[BR1], 0.520[CA1], 0.401[MZ1] 15 DYS4390.656 0.694[W1], 0.656[US2], 0.666[Cau3], 0.652[AA3], 0.0038 0.715[CN3],0.666[CN5], 0.600[KR1], 0.592[JP1], 0.688[PL1], 0.703[BY1], 0.668[ES1],0.628[ES2], 0.684[ES3], 0.655[ES4], 0.708[EU1], 0.636[NL1], 0.678[CZ1],0.679[BR1], 0.603[CA1], 0.612[MZ1], 0.620[ZA-E1], 0.720[ZA-I1],0.570[ZA-X1] 16 DYS444 0.666 0.592[US1], 0.620[Cau1], 0.650[AA1],0.610[US3], 0.0055 0.767[CN1], 0.756[CN5] 17 DYS447 0.742 0.781[W1],0.747[US2], 0.580[Cau1], 0.770[AA1], 0.0021 0.640[Cau2], 0.780[AA2],0.770[US3], 0.803[CN5], 0.776[EU1], 0.702[NL1], 0.719[BR1],0.871[ZA-I2], 0.679[ZA-E2], 0.774[ZA-X2] 18 DYS448 0.666 0.782[W1],0.721[US2], 0.550[Cau2], 0.710[AA2], 0.0004 0.73[US3], 0.602[Cau3],0.699[AA3], 0.745[CN2], 0.767[CN4], 0.704[CN5], 0.725[JP1], 0.462[BY1],0.585[EU1], 0556[NL1], 0.722[CZ1], 0.669[BR1], 0.588[ZA-I2],0.643[ZA-E2], 0.689[ZA-X2] 19 DYS449 0.831 0.874[W1], 0.832[US1],0.740[Cau1], 0.860[AA1], 0.0122 0.770[Cau2], 0.870[AA2], 0.840[US3],0.843[KR3], 0.857[EU1], 0.809[NL1], 0.877[CZ1], 0.831[BR1],0.780[ZA-E1], 0.880[ZA-I1], 0.780[ZA-X1], 0.867[ZA- I2], 0.787[ZA-E2],0.821[ZA-X2], 0.864[ZA1] 20 DYS456 0.660 0.706[W1], 0.700[US2],0.820[Cau2], 0.520[AA2], 0.0049 0.670[US3], 0699[CN2], 0.716[CN3],0.644[CN5], 0.474[JP1], 0.731[ES1], 0717[NL1], 0.796[Cz1], 0.691[BR1],0.622[ZA-I2], 0.728[ZA-E2], 0.434[ZA- X2] 21 DYS458 0.775 0.748[W1],0.765[US2], 0.810[Cau2], 0.750[AA2], 0.0084 0.821[CN2], 0.786[CN5],0.821[JP1], 0.748[BY1], 0.787[NL1], 0.791[BR1], 0.780[ZA-E1],0.780[ZA-I1], 0.690[ZA-X1] 22 DYS459 ab 0.694 0.647[US1], 0.67[Cau2],0.75[AA2], 0.75[US3], 0.0027 0.641[CN3], 0.704[NL1] 23 DYS460 0.5890.544[W1], 0.570[US2], 0.734[CN3], 0.699[CN5], 0.0062 0.563[PL1],0.607[BY1], 0.547[ES1], 0.624[ES2], 0.522[ES3], 0.577[ES4], 0.588[NL1],0.550[BR1], 0.560[CA1], 0.555[MZ1] 24 DYS471DYS610 0.873 0.844[Cau3],0.902[AA3] NA 25 DYS481 0.774 0.900[W2], 0.840[US3], 0.851[CN6],0.776[CN7], 0.0050 0.674[CN8], 0.752[CN9], 0.637[CN10], 0.860[JP1],0.840[EU1], 0.700[ZA-E1], 0.680[ZA-I1], 0.800[ZA- X1], 0.689[ZA-I2],0.712[ZA-E2], 0.816[ZA-X2], 0.851[ZA1] 26 DYS487 0.4550.62[W2]0.444[Cau3], 0.301[AA3] 0.0018 27 DYS488 0.239 0.58[W2],0.28[Cau2], 0.05[AA2], 0.23[US3], 0.0004 0.252[Cau3], 0.138[AA3],0.242[CN6], 0.283[CN7], 0.22[CN8], 0.081[CN9], 0.271[CN10] 28 DYS5040.717 0.810[US1], , 0.752[Cau3], 0.718[AA3], 0.735[JP1], 0.00320.672[ZA-I2], 0.674[ZA-E2], 0.661[ZA-X2] 29 DYS505 0.680 0.710[W2],0.667[US1], 0.680[US3], 0.744[JP1], 0.0015 0.599[EU1] 30 DYS508 0.6270.71[W2], 0.688[US1], 0.73[US3], 0.406[JP1], 0.0030 0.602[EU1] 31 DYS5180.848 0.870[W3], 0.800[ZA-E1], 0.850[ZA-I1], 0.870[ZA- 0.0184 X1],0.850[ZA-I2], 0.815[ZA-E2], 0.866[ZA-X2], 0.862[ZA1] 32 DYS522 0.6410.740[W2], 0.659[US1], 0.600[Cau1], 0.630[AA1], 0.0010 0.64[Cau2],0.590[AA2], 0.640[US3], 0.691[CN4], 0.582[JP1], 0.641[EU1], 33 DYS526 I*0.780 0.780[W3] 0.0027 34 DYS526 II* 0.880 0.880[W3] 0.0125 35 DYS527ab0.838 0.850[Cau1], 0.790[AA1], 0.850[Cau2], 0.780[AA2], NA 0.920[US3] 36DYS532 0.760 0.777[US1], 0.815[ZA-I2], 0.764[ZA-E2], 0.686[ZA- 0.0032X2] 37 DYS533 0.636 0.720[W2], 0.639[US1], 0.0050 0.620[US3], 0.566[EU1]38 DYS534 0.756 0.756[US1] 0.0065 39 DYS540 0.467 0.62[W2], 0.441[US1],0.340[JP1] 0.0033 40 DYS547 0.870 0.870[W3] 0.0236 41 DYS549 0.6440.720[W2], 0.0046 0.604[JP1], 0.608[EU1] 42 DYS552 0.621 0.729[ZA-I2],0.630[ZA-E2], 0.503[ZA-X2 0.0027 43 DYS557 0.737 0.774[US1], 0.60[Cau1],0.78[AA1], 0.66[Cau2], 0.0038 0.78[AA2], 0.79[US3], 0.734[CN5],0.72[ZA-E1], 0.86[ZA-I1], 0.67[ZA-X1] 44 DYS570 0.792 0.830[W3],0.860[W2], 0.784[US1], 0.780[Cau2], 0.810[AA2], 0.0124 0.790[US3],0.822[JP1], 0.804[EU1], 0.770[NL1], 0.773[BR1], 0.750[ZA-E1],0.820[ZA-I1], 0.700[ZA-X1] 45 DYS576 0.764 0.830[W3], 0.820[W2],0.797[US1], 0.790[Cau2], 0.0143 0.820[AA2], 0.830[US3], 0.802[CN6],0.544[CN7], 0.537[CN8], 0.770[CN9], 0.766[CN10], 0.771[JP1], 0.772[EU1],0.740[[NL1], 0.820[BR1], 0.778[ZA-I2], 0.752[ZA-E2], 0.746[ZA-X2] 46DYS607 0.701 0.63[Cau2], 0.72[AA2], 0.77[US3], 0.684[NL1], NA0.71[ZA-E1], 0.81[ZA-I1], 0.58[ZA-X1] 47 DYS612 0.810 0.850[W3],0.770[ZA-E1], 0.800[ZA-I1], 0.810[ZA-X1], 0.809[ZA-I2], 0.01450.764[ZA-E2], 0.829[ZA-X2], 0.849[ZA1] 48 DYS626 0.827 0.850[W3],0.850[ZA-E1], 0.800[ZA-I1], 0.790[ZA-X1], 0.819[ZA-I2], 0.01220.835[ZA-E2], 0.819[ZA-X2], 0.854[ZA1] 49 DYS627 0.860 0.860[US3] 0.012350 DYS635 0.702 0.739[US1], 0.718[CN5], 0.656[JP1], 0.677[BY1],0.758[BR1], 0.0039 0.600[ZA-E1], 0.820[ZA-I1], 0.650[ZA-X1] 51 DYS6430.745 0.820[W2], 0.755[US1], 0.780[US3], 0.0015 0.718[JP1], 0.650[EU1]52 DYS644 0.823 .91[ZA-E1], 0.91[ZA-I1], 0.80[ZA-X1], 0.783[ZA-I2],0.0032 0.718[ZA-E2], 0.763[ZA-X2], 0.875[ZA1] 53 DYS685 0.8230.786[Cau3], 0.859[AA3] NA 54 DYS688 0.895 0.89[Cau3], 0899[AA3] NA 55DYS703 0.543 .537[Cau3], 0.549[AA3] NA 56 DYS707 0.599 0.553[Cau3],0.644[AA3] NA 57 DYS710 0.831 0.71[ZA-E1], 0.75[ZA-I1], 0.75[ZA-X1],0.937[ZA-I2], NA 0.925[ZA-E2], 0.830[ZA-X2], 0.916[ZA1] 39 Y GATA H40.586 0.599[W1], 0.611[US2], 0.590[US3], 0.603[CN5], 0.572[JP1],0.558[PL1,] 0.0032 0.6561[BY1], 0.412[ES2], 0.604[ES3], 0.573[ES4],0.623[NL1], 0.581 [BR1], 0.549[MZ1], 0.656[ZA-I2], 0.588[ZA-E2],0.598[ZA1] *DYS526I/II is a multicopy marker similar to DYS389I/II, andnot included in the list of potential new markers.

TABLE 1A Population Background Key W1: World, N = 73, W2: World, N = 74,W3: World, N = 604, US1: USA, N = 660, Ref. (3) Ref. (4) Ref. (1) Ref.(5) US2: USA, N = 647, Cau1: USA-Caucasian, AA1: USA-African Cau2:USA-Caucasian, Ref. (6) N = 50, American, N = 100, N = 98, Ref. (7) Ref.(7) Ref. (8) AA2: USA-African Cau3: USA-Caucasian, AA3: USA-African US3:USA, N = 572, American, N = 51, N = 114, American, N = 110, Ref. (10)Ref. (8) Ref. (9) Ref. (9) CN1: China, N = 582, CN2: China, N = 108,CN3: China, N = 105, CN4: China-Han, Ref. (11) Ref. (12) Ref. (13) N =106, Ref. (14) CN5: China, N = 158, CN6: China-Daur, N = 38, CN7:China-Kazak, N = 39, CN8: China-Uighur, Ref. (15) Ref. (16) Ref. (16) N= 37, Ref. (16) CN9: China-Xibe, CN10: China-Kirgiz, KR1:Chinese-Korean, KR2: Korea, N = 316, N = 34, N = 26, N = 201, Ref. (18)Ref. (16) Ref. (16) Ref. (17) KR3: Korea, N = 301, JP1: Japan, N = 238,PL1: Poland, N = 208, BY1: Belarus, N = 328, Ref. (19) Ref. (20, 21)Ref. (22) Ref. (23) ES1: Spain, N = 768, ES2: Spain, N = 76, ES3: Spain,N = 134, ES4: Spain, N = 148, Ref. (24) Ref. (25) Ref. (26) Ref. (27)EU1: Europe NL1: Dutch & Belgian, CZ1: Czech-Caucasian, BR1: Brazil, N =873, (German, Dutch, N = 245, N = 50, Ref. (31) Turkish), N = 391, Ref.(29) Ref. (30) Ref. (28) CA1: Central Africa, MZ1: Mozambique, ZA-E1:South Africa- ZA-I1: South Africa- N = 101, (32) N = 112, English, N =101, Indian, N = 77, Ref. (33) Ref. (34) Ref. (34) ZA-X1: South Africa-ZA-I2: South Africa- ZA-E2: South Africa- ZA-X1: South Africa- Xhosa, N= 88, Indian, N = ?, English, N = ?, Xhosa, N = ?, Ref. (34) Ref. (35)Ref. (35) Ref. (35) ZA1: South Africa, N = 279, Ref. (36)

Mutation Rate.

Inclusion of a selection of one or more highly mutating Y-STR markers isalso considered. The individual mutation rates (MR) of Y-STR markers hasbeen described in US Application Publication 2011/0263437 A1, andBallantyne, et al, Forensic Sci Int. Genet. (2011),doi:10.1016/j.fsigen.2011.04.017). A rapidly mutating Y-STR marker mayhave a mutation rate greater than about 10⁻². TABLE 1 shows the mutationrate for each of the Y-STR markers considered for inclusion in themultiplex panels.

FIG. 1 shows Y-STR markers mapped for Gene Diversity on the x axis vs.mutation rate on the y axis. Diamonds represent Y-STR marker locipresently in the commercially Yfiler® multiplex analysis kit. Trianglesrepresent Y-STR markers not part of the Yfiler® kit. Y-STR markers to beconsidered for inclusion in the multiplex panel described here may beselected for a combination of gene diversity and mutation rateproperties, amongst other characteristics.

In devising a Y-STR multiplex assay panel that is separated and detectedusing electrophoresis, multiplex design can take advantage of a numberof differentiable fluorescent dye channels, and differing fragment sizeto place detection of each individual allele region for a marker at aselected portion of the electrophoretic run, permitting the detection oflabeled amplicons for all of the multiplexed loci without overlappingsignals. Available “space” within the multiplex, when incorporating allthe Yfiler® markers, can result in severe constraint in addingadditional Y-STR markers to the panel. This constraint may be reflectedin primer design consideration. In another aspect, the primer design andplacement may also need to provide for a separation of 1 or 2 base pairsbetween alleles of different Y-STR markers. In some embodiments, allelesof different Y-STR markers are separated by a 2 base pair “space” in theelectrophoretic analysis, permitting more accurate allele and markeridentification.

The use of mini-STRs, which produce amplicons of less than about 220 bp,also permit increased plexy while also permitting amplification and thusdetection of degraded DNA. However, not every STR marker can be adaptedfor use as a mini-STR. If the repeat sequence plus necessary flankingbases add up to too many bases or if the flanking sequence doesn't allowfor specific primer design, the STR marker may be unsuitable formini-STR use. The Y-STR markers that are unsuitable as mini-STR markersare limited in placement within the assay panel to a region reserved forlarger amplicon fragment detection. For example, some primers designedto place DYF404S1 and DYF387S1 at about 200 base pairs did not providesatisfactory results, and those markers may be more successfully placedin a multiplex panel at a larger amplicon size range.

Additional considerations when selecting Y-STR markers for inclusionwithin the multiplex panels described here are discussed further in thesection describing primer design, and include evaluation for specificity(i.e., only the target nucleic acid is amplified); sensitivity (i.e.,even a small amount of a target nucleic acid within a mixed forensicsample can be accurately amplified), and for potential undesirableinteractions within the multiplex.

Some Y-STR markers may not yield the highest probability of successfulincorporation into a multiplex assay panel, based upon initial studies.For example, 3 different primer pairs were tested for DYS626. Two of theprimer pairs showed additional peaks in samples with 1:500 male/femaleDNA, while the third primer pair showed interactions with Yfiler®markers DYS392 and DYS439. In another example, two different primerpairs for DYF404S1 were tested. Both had only ⅓ of their original peakheight when used in 1:500 male/female DNA samples. This may lead to adrop out problem in casework samples. In a third example, two differentprimers were tested for DYS547, but both displayed undesirableamplification of female DNA when tested in a 1:500 male:female mixture.All of these markers may be successfully used in multiplex panels ifforensic samples including male:female mixtures were not used for sourceDNA. For instance, if the Y-STR multiplex panel is used to discriminatebetween male relatives, any of these markers may be successful additionsto an improved Y-STR multiplex panel compared to currently commerciallyavailable kits.

Another complication may arise when evaluating markers such as DYS612,which due to the nature of its trinucleotide repeat, may provideamplified products which include significant percentages of stutter.Stutter is an artifact seen when amplifying short tandem repeats and mayoccur at one repeat unit shorter in length than the parent allele. Inforensic analysis, stutter complicates the analysis of DNA profiles frommultiple contributors, so a marker with pronounced tendency for stuttermay not be as desirable for use in multiplex forensic panels used forsamples having mixed sources, potentially at significantly varyingratios. It may be used successfully, however, in analysis for resolutionof individual male identity/relatedness.

In some cases, the primers for the current Y-Filer® markers may bealtered to remove interactions with the newly added Y-STR markers of thevarious panels.

Several different multiplex panels are shown here, which include thecurrent Y-Filer® markers plus additional Y-STR markers to maximizehaplotype resolution.

FIG. 2 shows a schematic representation of Panel 1, a 35 plex Y-STRmultiplex assay panel. Six dyes are used, including a sizestandard/allelic ladder labeled with a sixth differentiable fluorescentdye (not shown). Thirteen markers are mini-STRs and the entire panel iscaptured in an electrophoretic run of about 450 base pairs.

FIG. 3 shows a schematic representation of Panel 2, a 27-plex Y-STRmultiplex assay panel that includes all Yfiler® STR markers and 10additional Y-STR markers (including a double copy marker) having a GeneDiversity value greater than 0.75, where the multiplex includes 7rapidly mutating Y-STR markers. Six dyes are used, including a sizestandard/allelic ladder labeled with a sixth differentiable fluorescentdye (not shown). Twelve markers are mini Y-STR markers, and the entirepanel is captured in an electrophoretic run of about 370 base pairs.

FIG. 4 shows a schematic representation of Panel 3, a 30-plex Y-STRmultiplex assay panel that includes all Yfiler® STR markers, and 9additional (10, including the double marker) Y-STR markers having a GeneDiversity value greater than 0.75. Six dyes are used, including a sizestandard/allelic ladder labeled with a sixth differentiable fluorescentdye (not shown). Twelve markers are mini-STR markers, and the entirepanel is captured in an electrophoretic run of about 420 base pairs.

FIG. 5 shows a schematic representation of Panel 4, a 27-plex Y-STRmultiplex containing all Yfiler® Y-STR markers, the three best markersbased on average Gene Diversity value, and six additional markers. Sixdyes are used, including a size standard/allelic ladder labeled with asixth differentiable fluorescent dye (not shown). Twelve markers aremini-STR markers, and the entire panel is captured in an electrophoreticrun of about 370 base pairs.

FIG. 6 shows a schematic representation of Panel 5, a 27-plex Y-STRmultiplex containing all Yfiler® Y-STR markers, and 12 other Y-STRmarkers. Six dyes are used, including a size standard/allelic ladderlabeled with a sixth differentiable fluorescent dye (not shown). Twelvemarkers are mini-STR markers, and the entire panel is captured in anelectrophoretic run of about 410 base pairs.

For the Y-STR multiplex assay panels of FIGS. 3-6, an additional marker,DYS460, which has a moderate Gene Diversity value, could be included inDye 2 channel, to obtain a total of 13 mini-STR markers.

FIG. 7 shows a schematic representation of Panel 6, a 27-plex Y-STRmultiplex containing all Yfiler® Y-STR markers and ten additionalmarkers. Two of the twenty seven markers are double copy markers (DYS385ab and DYF387S1ab), and eleven markers are mini-markers of less thanabout 220 base pairs. The at least 11 Y-STR markers having a size ofless than about 220 base pairs may be DYS576, DYS389I, DYS460, DYS458,DYS19, DYS456, DYS390, DYS570, DYS437, DYS393, and DYS439. Six dyes areused, including a size standard/allelic ladder labeled with a sixthdifferentiable fluorescent dye (not shown). At least five markers arerapidly mutating markers (see TABLE 1), and represent six total rapidlymutating markers in the panel because DYF387S1ab is a double copymarker. The rapidly mutating Y-STR markers may be DYF387S1ab, DYS449,DYS570, DYS576, and DYS627. DYS446 was not included in this panelbecause its proximity to the DYS393 marker (present in the Yfiler®multiplex) may cause artifacts from amplification through both markers.The entire panel is captured in a range of about 76-400 base pairs uponelectrophoretic separation. FIG. 9 shows allele ranges for Panel 6 incomparison to database and other Y-STR panels.

FIG. 8 shows a graphical representation of an electrophoretic separationusing the 27-plex Y-STR marker panel of FIG. 7 (Panel 6), showing eachdifferentiable dye channel in a separate lane and not showing the sizestandard dye channel using the sixth dye. In TABLE 2, entries 1-18 and20-26 describe the individual markers, the repeat sequences, the repeattypes, and the chromosomal location of the Y-STR markers of Panel 6.Panel 6 is a multiplex assay for the amplification of a set of primersfor the amplification of DYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II,DYS390, DYS391, DYS392, DYS393, and DYS460, DYS437, DYS438, DYS439,DYS448, DYS449, DYS456, DYS458, DYS481, DYS533, DYS570, DYS576, DYS627,DYS635, DYS643, and Y-GATA-H4.

FIG. 10 shows a schematic representation of Panel 7, a 27-plex Y-STRmultiplex containing all Yfiler® Y-STR markers and ten additionalmarkers. Two of the twenty seven markers are double copy markers(DYS385ab and DYF387S1ab), and eleven markers are mini-markers of lessthan about 220 base pairs. The at least 11 Y-STR markers having a sizeof less than about 220 base pairs may be DYS576, DYS389I, DYS460,DYS458, DYS19, DYS456, DYS390, DYS570, DYS437, DYS393, and DYS439. Sixdyes are used, including a size standard/allelic ladder labeled with asixth differentiable fluorescent dye (not shown). At least five markersare rapidly mutating markers (see Table 1), and represent a total ofseven rapidly mutating markers in the panel because DYF387S1ab is adouble copy marker. The rapidly mutating Y-STR markers may beDYF387S1ab, DYS449, DYS518, DYS570, DYS576, and DYS627. The entire panelmay be captured in a range of about 68 to about 406 base pairs uponelectrophoretic separation. FIG. 12 shows allele ranges for Panel 7 incomparison to database and other Y-STR panels.

FIG. 11 shows a graphical representation of an electrophoreticseparation using the 27-plex Y-STR marker panel of FIG. 10 (Panel 7),showing each differentiable dye channel in a separate lane and notshowing the size standard dye channel using the sixth dye. In TABLE 2,entries 1-24 and 26 describe the individual markers, the repeatsequences, the repeat types, and the chromosomal location of the Y-STRmarkers of Panel 7. Panel 7 is a multiplex assay for the amplificationof DYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391,DYS392, DYS393, DYS460, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456,DYS458, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627, DYS635, andY-GATA-H4.

TABLE 2Y-STR markers, including repeat sequence, type, and chromosomal location.Repeat Chr. Marker Repeat Sequence type Location Notes  1 DYF387S1ab[AAAG]3[GTAG]1[GAAG]4- 4 Yq12 Yq11.23 a. [AAAG]2[GAAG]1-[AAAG]2[GAAG]m[AAAG]n (SEQ ID NO: 8)  2 DYS19 [TAGA]3TAGG[TAGA]n 4Yp11.2 (SEQ ID NO: 9)  3 DYS385ab [GAAA]n 4 Yq11.222  4 DYS389I[TCTG]3[TCTA]n 4 Yq11.221 (SEQ ID NO: 10)  5 DYS389II [TCTG]m[TCTA]o- 4Yq11.221 [TCTG]3[TCTA]n (SEQ ID NO: 11)  6 DYS390 [TCTG]8[TCTA]n- 4Yq11.221 [TCTG]1[TCTA]4 (SEQ ID NO: 12)  7 DYS391 [TCTA]n 4 Yq11.21  8DYS392 [TAT]n 3 Yq11.223  9 DYS393 [AGAT]n 4 Yq12 10 DYS437[TCTA]n[TCTG]2[TCTA]4 4 Yq11.221 (SEQ ID NO: 13) 11 DYS438 [TTTTC]n 5Yq11.221 12 DYS439 [GATA]n 4 Yq11.221 13 DYS448 [AGAGAT]nN42-[AGAGAT]m 6Yq11.223 (SEQ ID NO: 14) 14 DYS449 [TTCT]nN22[TTCT]3- 4 Yp11.2 b.N12[TTCT]m (SEQ ID NO: 15) 15 DYS456 [AGAT]n 4 Yp11.2 16 DYS458 [GAAA]n4 Yp11.2 17 DYS460 [ATAG]n 4 Yq11.222 b. 18 DYS481 [CTT]n 3 Yp11.2 b. 19DYS518 [AAAG]3[GAAG]1[AAAG]n- 4 Yq11.221 b. [GGAG]1- [AAAG]4N6[AAAG]m-N27[AAGG]4 (SEQ ID NO: 16) 20 DYS533 [ATCT]n 4 Yq11.221 b. 21 DYS570[TTTC]n 4 Yp11.2 b. 22 DYS576 [AAAG]n 4 Yp11.2 b. 23 DYS627[AGAA]3N16[AGAG]3- 4 Yp11.2 c. [AAAG]nN82[AAGG]3 (SEQ ID NO: 17) 24DYS635 [TCTA]4[TGTA]2- 4 Yq11.221 [TCTA]2[TGTA]2-[TCTA]2[TGTA]0,2[TCTA]n 25 DYS643 [CTTTT]n 5 Yq11.221 b. (SEQ ID NO: 18)26 GATA-H4 [TAGA]nN12[GAT

]2- 4 Yq11.221 d. AA[TAGA]4 (SEQ ID NO: 19) ii DYS549 [GATA]n 4 Yq11.223b. TABLE LEGEND: variable repeats in BOLD, Different Sequence listed inItalics and underlined. a. Sequence Repeat confirmed through in-housesequencing. Ballantyne et al. 2011 published different repeat structure:AAAG]3[GTAG][GAAG]4N16-[GAAG]9[AAAG]n, and different chromosomallocation: Yq11.2 Yq11.23. b. Sequence Repeat confirmed through in-housesequencing. c. Sequence Repeat confirmed through in-house sequencing.Ballantyne et al. 2011 published different repeat structure:AGAA]3N16[AGAG]3[AAAG]n N81 [AAGG]3 d. Sequence differs in NCBI RefSeqNC000024 from STRbase: [TAGA]12N12[GAT G ]2AA[TAGA)4.

Alleles.

Numerous new alleles have been reported, and may be incorporated intothe Y-STR marker analysis. FIG. 9 shows the alleles for each marker forPanel 6, in comparison to other panels. FIG. 12 shows the alleles foreach marker of Panel 7. Both panels contain expanded allele ranges forselected markers. The allelic ladder for Panels 6 and 7 incorporatethese new alleles. For example the expanded allele ranges are shown inFIG. 13 for marker DYS438. The upper lane shows the alleles from theYfiler® kit (8-13 for a total of 6) while the lower lane shows theexpanded 6-16 repeat range for the Y-STR Panels 1-7, which also includesvirtual alleles 5, 8.2 (identified in the schematic as 8), and 17.

Primer Design.

Once a set of loci for co-amplification in a single multiplex reactionis identified, one can determine primers suitable for co-amplifying eachlocus in the set. Oligonucleotide primers may be added to the reactionmix and serve to demarcate the 5′ and 3′ ends of an amplified DNAfragment. One oligonucleotide primer anneals to the sense (+) strand ofthe denatured template DNA, and the other oligonucleotide primer annealsto the antisense (−) strand of the denatured template DNA. Typically,oligonucleotide primers may be approximately 12-25 nucleotides inlength, but their size may vary considerably depending on suchparameters as, for example, the base composition of the templatesequence to be amplified, amplification reaction conditions, etc.Oligonucleotide primers can be designed to anneal to specific portionsof DNA that flank a locus of interest, to specifically amplify theportion of DNA between the primer-complementary sites. The length of theprimer may need to be modified in order to be more specific and preventamplification of non-target nucleic acid. For example, it was discoveredthat lengthening the primer for DYS456 marker resolved a problem withunwanted amplification of female DNA for a subset of female DNA sampleswhen amplifying a mixed male:female sample. On the left hand side ofFIG. 14, two partial electrophoretic separation segments showing theDYS456 marker demonstrate inappropriate amplification of female DNA(off-scale peak at left side of the separation segment) when the selectfemale A and female B samples were present in a 0.5 ng male/250 ngfemale sample and using standard length (25 nucleotides or less)primers. In contrast, using lengthened primers (28 nucleotides long) toamplify DYS456, when analyzing these two problematic male/femalemixtures, as shown in the right hand panels of FIG. 14, appropriatelyamplified male alleles are seen for this Y-STR marker (on-scale solopeak with no evidence of inappropriate female DNA amplification).

Oligonucleotide primers may include adenosine, thymidine, guanosine, andcytidine, as well as uracil, nucleoside analogs (for example, but notlimited to, inosine, locked nucleic acids (LNA), non-nucleotide linkers,peptide nucleic acids (PNA) and phosporamidites) and nucleosidescontaining or conjugated to chemical moieties such as radionuclides(e.g., ³²P and ³⁵S), fluorescent molecules, minor groove binders (MGBs),or any other nucleoside conjugates known in the art.

Generally, oligonucleotide primers can be chemically synthesized. Careshould be taken in selecting the primer sequences used in the multiplexreaction. Inappropriate selection of primers may produce undesirableeffects such as a lack of amplification, amplification at one site ormultiple sites besides the intended target locus, primer-dimerformation, undesirable interactions between primers for different loci,production of amplicons from alleles of one locus which overlap (e.g.,in size) with alleles from another locus, or the need for amplificationconditions or protocols particularly suited for each of the differentloci, which conditions/protocols are incompatible in a single multiplexsystem. Primers can be developed and selected for use in the multiplexsystems of this teaching by, for example, employing a re-iterativeprocess of multiplex optimization that is well familiar to one ofordinary skill in the art: selecting primer sequences, mixing theprimers for co-amplification of the selected loci, co-amplifying theloci, then separating and detecting the amplified products to determineeffectiveness of the primers in amplification.

Primers can be selected by the use of any of various software programsavailable and known in the art for developing amplification and/ormultiplex systems. See, e.g., Primer Express® software (AppliedBiosystems, Foster City, Calif.). In the example of the use of softwareprograms, sequence information from the region of the locus of interestcan be imported into the software. The software then uses variousalgorithms to select primers that best meet the user's specifications.

Initially, this primer selection process may produce any of theundesirable effects in amplification described above, or an imbalance ofamplification product, with greater product yield for some loci than forothers because of greater binding strength between some primers andtheir respective targets than other primers, for example resulting inpreferred annealing and amplification for some loci. Or, the primers maygenerate amplification products which do not represent the target locialleles themselves; i.e., non-specific amplification product may begenerated. These extraneous products resulting from poor primer designmay be due, for example, to annealing of the primer with non-targetregions of sample DNA, or even with other primers, followed byamplification subsequent to annealing.

When imbalanced or non-specific amplification products are present inthe multiplex systems during primer selection, individual primers can betaken from the total multiplex set and used in amplification withprimers from the same or other loci to identify which primers contributeto the amplification imbalance or artifacts. Once two primers whichgenerate one or more of the artifacts or imbalance are identified, oneor both contributors can be modified and retested, either alone in apair, or in the multiplex system (or a subset of the multiplex system).This process may be repeated until product evaluation results inamplified alleles with no amplification artifacts or an acceptable levelof amplification artifacts in the multiplex system.

The optimization of primer concentration can be performed either beforeor after determination of the final primer sequences, but most often maybe performed after primer selection. Generally, increasing theconcentration of primers for any particular locus increases the amountof product generated for that locus. However, primer concentrationoptimization is also a re-iterative process because, for example,increasing product yield from one locus may decrease the yield fromanother locus or other loci. Furthermore, primers may interact with eachother, which may directly affect the yield of amplification product fromvarious loci. In sum, a linear increase in concentration of a specificprimer set may not necessarily equate with a linear increase inamplification product yield for the corresponding locus. Reference ismade to Simons, U.S. Pat. No. 5,192,659, for a more detailed descriptionof locus-specific primers, the teaching of which is incorporated hereinby reference in its entirety.

Locus-to-locus amplification product balance in a multiplex reaction mayalso be affected by a number of parameters of the amplificationprotocol, such as, for example, the amount of template (sample DNA)input, the number of amplification cycles used, the annealingtemperature of the thermal cycling protocol, and the inclusion orexclusion of an extra extension step at the end of the cycling process.An absolutely even balance in amplification product yield across allalleles and loci, although theoretically desirable, is generally notachieved in practice.

Mobility Modifiers.

In some embodiments the electrophoretic mobility of the amplificationproduct, i.e., amplicon containing the STR for a given locus, can beadjusted to avoid overlapping with the electrophoretic mobility range ofanother, different STR locus amplicon. This can be done in at least twodifferent approaches that may be used in isolation or in combinationwith one another. In one approach, the position of the primers isadjusted to create either a smaller or larger amplification product toavoid overlapping the molecular weight size of another locus duringelectrophoresis. In another approach, mobility modifier moietiesincluding, but not limited to, for example, polyethyleneoxide repeatsubunits optionally interrupted by phosphodiester or phosphotriestersubunits may be incorporated as a non-nucleotide linker between afluorescent dye at the 5′-end of the primer and the primer sequence. Thepolyethyleneoxide repeat subunits may be triethyleneoxide,tetraethyleneoxide, pentaethyleneoxide, hexaethyleneoxide (HEO),heptaethyleneoxide, or octaethyleneoxide subunits. The polyethylenerepeat subunit may be repeated once, twice, three, four, five, six,seven, eight, nine, ten, eleven or twelve times. The mobility modifiermoiety may be attached to the 5′-end of the primer sequence via aphosphodiester moiety. See, for example, U.S. Pat. Nos. 5,470,705;5,514,543; 5,580,732; 5,624,800; 5,703,222; 5,777,096; 5,807,682;5,989,871; 6,395,486; 6,734,296; 6,756,204; 6,743,905; 7,074,569;7,115,376; 7,129,050; and 7,897,338, each of which is incorporated byreference herein in its entirety. Similarly, the amplification primersmay contain additional nucleotides (at the 5′ end) that do not hybridizeto the locus, but are added to create the desired mobility of theamplicon for the detection method employed, e.g., electrophoresis ormass spectroscopy. The resulting PCR amplification product (of thelarger of the two PCR products) contains the mobility modifiermolecules, increasing the molecular weight of the PCR product and thus aperceived shift in the molecular weight of the larger PCR product to aneven larger size. The molecular weight range, which is correlated to thesize of an amplicon, therefore is apparently higher when the ampliconincludes a mobility modifier moiety.

When amplicons of a Y-STR marker have a base pair size of less thanabout 220 base pairs, it may be referred to herein as a mini-Y-STRmarker. In some embodiments, each of the amplicons of at least 11 Y-STRmarkers has a base pair size of less than about 220 base pairs. In someembodiments, each of the amplicons of at least one of at least 11 Y-STRmarkers having an effective base pair size of less than about 220 basepairs also includes a mobility modifier moiety. When the size for eachof the amplicons of the mini Y-STR markers is stated to be less thanabout 220 base pairs, then the size range of less than about 220 basepairs includes 80-90% of the alleles for the mini Y-STR marker. In someembodiments, the at least 11 mini Y-STR markers may include DYS576,DYS389I, DYS460, DYS458, DYS19, DYS456, DYS390, DYS570, DYS437, DYS393,and DYS439. In other embodiments, the at least 11 mini Y-STR markers mayinclude DYS19, DYS458, DYS456, DYS505, DYS481, DYS460, DYS437, DYS389I,DYS576, DYS390, DYS570, DYS391, and DYS393. In yet other embodiments,the at least 11 mini Y-STR markers may include DYS19, DYS458, DYS456,DYS439, DYS481, DYS437, DYS389I, DYS576, DYS390, DYS570, DYS391, andDYS393. In some other embodiments, at least 11 mini Y-STR markers mayinclude DYS19, DYS458, DYS456, DYS439, DYS481, DYS437, DYS389I, DYS576,DYS390, DYS570, DYS391, and DYS393. In some embodiments, the at least 11mini Y-STR markers are selected from DYS576, DYS389I, DYS460, DYS458,DYS19, DYS456, DYS390, DYS570, DYS437, DYS393, DYS439, DYS505, DYS481,and DYS391. In some embodiments, more than 11 mini-STR markers areselected from DYS576, DYS389I, DYS460, DYS458, DYS19, DYS456, DYS390,DYS570, DYS437, DYS393, DYS439, DYS505, DYS481, and DYS391. In someembodiments, the amplification primer for more than one Y-STR marker hasa mobility modifier moiety incorporated therein. When the amplificationprimers for more than one Y-STR marker have mobility modifier moieties,then the structure of the mobility modifier moiety of the amplificationprimer for each different Y-STR marker may be selected independently. Insome embodiments, more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15Y-STR markers have mobility modified amplification primers. In someembodiments, mobility modified amplification primers may be selected foramplifying Y-STR markers from the group including DYS627, DYS389II,DYS635, DYS389I, DYS391, DYS448, Y-GATA-H4, DYS19, DYS438, DYS390, andDYS449, in any combination or subcombination.

In some embodiments, a set of amplification primers including primersfor the amplification of at least 11 Y-STR markers is provided where theprimers are configured to provide each set of amplicons of the at least11 Y-STR markers having a base pair size less than about 220 base pairs.In some embodiments, detection of amplicon base pair size may beperformed by a fluorescence detection technique. In some embodiments,detection of amplicon base pair size may be performed by amobility-dependent analytical technique. The mobility-dependentanalytical technique may be capillary electrophoresis. In some otherembodiments, detection of the amplicon base pair size may be performedby a sequencing technique using no fluorescent dye labels. In someembodiments, the set of amplification primers may further includeprimers for the amplification of at least 5 additional Y-STR markerswhere the primers are configured to provide each set of amplicons of theat least 5 additional Y-STR markers having a base pair size greater thanabout 220 base pairs. In various embodiments, when the set ofamplification primers amplify more than 11 Y-STR markers, then the setof amplification primers may be configured to provide all of the sets ofamplicons of the more than 11 Y-STR markers having a base pair size lessthan about 410 base pairs. In various embodiments, when the set ofamplification primers amplify more than 11 Y-STR markers, then the setof amplification primers may be configured to provide all of the sets ofamplicons of the more than 11 Y-STR markers having a base pair size lessthan about 420 base pairs. The amplification primer set may include 25Y-STR markers. In some embodiments, the set of amplification primers islabeled with one of at least 5 fluorescent dyes. In some embodiments,the set of amplification primers may be configured to provide each setof the amplicons of the at least 11 Y-STR markers labeled with one of atleast 5 fluorescent dyes. The at least 5 fluorescent dyes used to labelthe primers and/or the amplicons may be configured to be spectrallydistinct. The set of amplification primers may further include at leastone amplification primer that includes a mobility modifier. The set ofamplification primers for the amplification of at least 11 Y-STR markersmay be configured to provide at least one set of amplicons of the Y-STRmarkers including a mobility modifier. In some embodiments, the set ofamplification primers amplifying at least 11 Y-STR markers, may amplifyDYS576, DYS389I, DYS460, DYS458, DYS19, DYS456, DYS390, DYS570, DYS437,DYS393, and DYS439. In other embodiments, the set of amplificationprimers amplifying the at least 11 Y-STR markers configured to provideeach set of amplicons of the at least 11 Y-STR markers having a basepair size less than about 220 base pairs, may amplify at least 5 Y-STRmarkers which are rapidly mutating loci. In some embodiments, the atleast 5 rapidly mutating Y-STR markers may include DYF387S1ab, DYS449,DYS570, DYS576, and DYS627. In other embodiments, the at least 5 rapidlymutating Y-STR markers may further include DYS518. In some embodiments,the set of primers for the amplification of at least 11 Y-STR markersmay be a set of primers for the amplification of DYF387S1ab, DYS19,DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS460,DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS481, DYS518,DYS533, DYS570, DYS576, DYS627, DYS635, and Y-GATA-H4. In otherembodiments, the set of primers for the amplification of at least 11Y-STR markers may be a set of primers for the amplification ofDYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392,DYS393, DYS460, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458,DYS481, DYS533, DYS570, DYS576, DYS627, DYS635, DYS643, and Y-GATA-H4.

In some embodiments, when primers for the amplification of more than the11 Y-STR markers are provided, then the primers are configured toprovide sets of amplicons of the more than 11 Y-STR markers having abase pair size less than about 400 base pairs. In some embodiments, whenprimers for the amplification of more than the 11 Y-STR markers areprovided, then the primers are configured to provide sets of ampliconsof the more than 11 Y-STR markers having a base pair size less thanabout 405 base pairs. In some embodiments, when primers for theamplification of more than the 11 Y-STR markers are provided, then theprimers are configured to provide sets of amplicons of the more than 11Y-STR markers having a base pair size less than about 410 base pairs. Insome embodiments, when primers for the amplification of more than the 11Y-STR markers are provided, then the primers are configured to providesets of amplicons of the more than 11 Y-STR markers having a base pairsize less than about 415 base pairs. In some embodiments, when primersfor the amplification of more than the 11 Y-STR markers are provided,then the primers are configured to provide sets of amplicons of the morethan 11 Y-STR markers having a base pair size less than about 420 basepairs. In some embodiments, when primers for the amplification of morethan the 11 Y-STR markers are provided, then the primers are configuredto provide all sets of amplicons of the more than 11 Y-STR markershaving a base pair size less than about 405 base pairs. In someembodiments, when primers for the amplification of more than the 11Y-STR markers are provided, then the primers are configured to provideall sets of amplicons of the more than 11 Y-STR markers having a basepair size less than about 410 base pairs. In some embodiments, whenprimers for the amplification of more than the 11 Y-STR markers areprovided, then the primers are configured to provide all sets ofamplicons of the more than 11 Y-STR markers having a base pair size lessthan about 415 base pairs. In some embodiments, when primers for theamplification of more than the 11 Y-STR markers are provided, then theprimers are configured to provide all sets of amplicons of the more than11 Y-STR markers having a base pair size less than about 425 base pairs.

In some embodiments, primers for the amplification of more than the 11Y-STR markers are provided wherein each of the amplicons of at least 11Y-STR markers has a base pair size of less than about 220 base pairs,and wherein the primers are configured to provide all sets of ampliconsof the more than 11 Y-STR markers having a base pair size less thanabout 400 base pairs. In some embodiments, primers for the amplificationof more than the 11 Y-STR markers are provided wherein each of theamplicons of at least 11 Y-STR markers has a base pair size of less thanabout 220 base pairs, and wherein the primers are configured to provideall sets of amplicons of the more than 11 Y-STR markers having a basepair size less than about 410 base pairs. In some embodiments, primersfor the amplification of more than the 11 Y-STR markers are providedwherein each of the amplicons of at least 11 Y-STR markers has a basepair size of less than about 220 base pairs, and wherein the primers areconfigured to provide all sets of amplicons of the more than 11 Y-STRmarkers having a base pair size less than about 415 base pairs. In someembodiments, primers for the amplification of more than the 11 Y-STRmarkers are provided wherein each of the amplicons of at least 11 Y-STRmarkers has a base pair size of less than about 220 base pairs, andwherein the primers are configured to provide all sets of amplicons ofthe more than 11 Y-STR markers having a base pair size less than about420 base pairs.

In yet another aspect, a set of amplification primers including primersfor the amplification of at least 22 Y-STR markers is provided, where atleast 5 of the Y-STR markers are rapidly mutating loci. In someembodiments, the at least 5 rapidly mutating Y-STR markers includeDYF387S1ab, DYS449, DYS570, DYS576, and DYS627. In some embodiments, theat least 5 rapidly mutating Y-STR markers include DYS518. In variousembodiments, the set of amplification primers configured to amplify theat least 22 Y-STR markers may be further configured to provide each setof amplicons of at least 11 Y-STR markers having a base pair size lessthan about 220 base pairs. In other embodiments, the set ofamplification primers for the amplification of at least 22 Y-STR markersmay be configured to provide sets of amplicons for the at least 22 Y-STRmarkers each having a base pair size of less than about 410 base pairs.In other embodiments, the set of amplification primers for theamplification of at least 22 Y-STR markers may be configured to providesets of amplicons for the at least 22 Y-STR markers each having a basepair size of less than about 420 base pairs. In some embodiments,detection of amplicon base pair size may be performed by fluorescencedetection. In some embodiments, detection of amplicon base pair size maybe performed by a mobility-dependent analytical technique. Themobility-dependent analytical technique may be capillaryelectrophoresis. In some other embodiments, detection of the ampliconbase pair size may be performed by a sequencing technique using nodetection of fluorescent dye labels. The amplification primer set mayinclude 25 Y-STR markers. In some embodiments, the set of amplificationprimers is labeled with one of at least 5 fluorescent dyes. In someembodiments, each set of the amplicons of the at least 22 Y-STR markersis labeled with one of at least 5 fluorescent dyes. The at least 5fluorescent dyes used to label the primers and/or the amplicons may beconfigured to be spectrally distinct. The set of amplification primersmay further include at least one amplification primer that includes amobility modifier. The set of amplification primers for theamplification of at least 22 Y-STR markers may be configured to provideat least one set of amplicons of the Y-STR markers where the at leastone set of amplicons includes a mobility modifier. In some embodiments,the at least 22 Y-STR markers may include DYF387S1ab, DYS19, DYS385ab,DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438,DYS439, DYS448, DYS449, DYS456, DYS458, DYS570, DYS576, DYS627, DYS635,and Y-GATA-H4. In other embodiments, the at least 22 Y-STR markers mayinclude DYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391,DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458,DYS518, DYS570, DYS576, DYS627, DYS635, and Y-GATA-H4.

Fluorophore Labeling.

In some embodiments of the present teaching, a fluorophore can be usedto label at least one primer of the multiplex amplification, e.g. bybeing covalently bound to the primer, thus creating a fluorescentlabeled primer. In some embodiments, primers for different target lociin a multiplex can be labeled with different fluorophores, eachfluorophore producing a different colored product depending on theemission wavelength of the fluorophore. These variously labeled primerscan be used in the same multiplex reaction, and their respectiveamplification products subsequently analyzed together. Either theforward or reverse primer of the pair that amplifies a specific locuscan be labeled, although the forward may more often be labeled. In someembodiments, the 5′ end of the forward primer may be labeled with afluorophore. When the primer is labeled at the 5′ end, it may befluorescently labeled through a linker to the 5′ phosphate. In otherembodiments, when is labeled at the 5′ end, it may be fluorescentlylabeled via a linker to the nucleobase.

The following are some examples of fluorophores well known in the artand suitable for use in the present teachings. The list is intended tobe exemplary and is by no means exhaustive. Some possible fluorophoresinclude: fluorescein (FL), which absorbs maximally at 492 nm and emitsmaximally at 520 nm; N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA™),which absorbs maximally at 555 nm and emits maximally at 580 nm;5-carboxyfluorescein (5-FAM™), which absorbs maximally at 495 nm andemits maximally at 525 nm;2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE™), whichabsorbs maximally at 525 nm and emits maximally at 555 nm;6-carboxy-X-rhodamine (ROX™), which absorbs maximally at 585 nm andemits maximally at 605 nm; CY3™, which absorbs maximally at 552 nm andemits maximally at 570 nm; CY5™, which absorbs maximally at 643 nm andemits maximally at 667 nm; tetrachloro-fluorescein (TET™), which absorbsmaximally at 521 nm and emits maximally at 536 nm; andhexachloro-fluorescein (HEX™), which absorbs maximally at 535 nm andemits maximally at 556 nm; NED™, which absorbs maximally at 546 nm andemits maximally at 575 nm; 6-FAM™, which emits maximally atapproximately 520 nm; VIC® which emits maximally at approximately 550nm; PET® which emits maximally at approximately 590 nm; and LIZ™, whichemits maximally at approximately 650 nm. See SR Coticone et al., U.S.Pat. No. 6,780,588; AmpFISTR® Identifiler™ PCR Amplification Kit User'sManual, pp. 1-3, Applied Biosystems (2001). Note that the above listedemission and/or absorption wavelengths are typical and can be used forgeneral guidance purposes only; actual peak wavelengths may vary fordifferent applications and under different conditions. Additionalfluorophores can be selected for the desired absorbance and emissionspectra as well as color as is known to one of skill in the art and areprovided in TABLE 3.

TABLE 3 Commercially Available Dyes. Fluorophore Abs (nm) Em (nm)Fluorophore Abs (nm) Em (nm) Methoxycoumarin 340 405 Dansyl 340 520Pyrene 345 378 Alexa Fluor ® 350 346 442 CF ™ 350 347 448 AMCA 349 448DyLight 350 353 432 Marina Blue ® dye 365 460 Dapoxyl ® dye 373 551Dialkylamino-coumarin 375 435 470-475 Bimane 380 458 SeTau 380 381 480Hydroxycoumarin 385 445 ATTO 390 390 479 Cascade Blue ® dye 400 420Pacific Orange ® dye 400 551 DyLight ® 405 400 420 Alexa Fluor ® 405 402421 SeTau 404 402 518 Cascade Yellow ® dye 402 545 CF ™ 405S 404 431CF ™ 405M 408 452 Pacific Blue ™ dye 410 455 PyMPO 415 570 DY-415 415467 SeTau 425 425 545 Alexa Fluor ® 430 434 539 ATTO 425 436 484 ATTO465 453 508 NBD 465 535 Seta 470 469 521 CF ™ 485 470-488 513 DY-485XL485 560 CF ™ 488A 490 515 DyLight ® 488 493 518 DY 496 493 521Fluorescein 494 518 ATTO 495 495 527 Alexa Fluor ® 488 495 519 OregonGreen ® 488 496 524 BODIPY ® 493/503 500 506 CAL Fluor ® Green 520 500522 DY-480XL 500 630 ATTO 488 501 523 Rhodamine Green dye 502 527BODIPY ® FL 505 513 DY505 505 530 DY 510XL 509 5902′,7′-Dichlorofluorescein 510 532 Oregon Green ® 514 511 530 DY-481XL515 650 ATTO 520 516 538 Alexa Fluor ® 514 518 540 CAL Fluor ® Gold 540519 537 DY 520XL 520 664 4′,5′-Dichloro- 2′,7′- 522 550 dimethoxy-fluorescein (JOE) DY -521XL 523 668 Eosin 524 544 Rhodamine 6G 525 555BODIPY ® R6G 528 550 Alexa Fluor ® 532 531 554 ATTO 532 532 553 BODIPY ®530/550 534 554 CAL Fluor ® Orange 560 534 556 DY-530 539 561 BODIPY ®TMR 542 574 DY-555 547 572 DY556 548 573 Quasar ® 570 548 570 Cy 3 550570 CF ™555 550 570 DY-554 551 572 DY 550 553 578 ATTO 550 554 576Tetramethyl- 555 580 Alexa Fluor ® 555 555 565 rhodamine (TMR) Seta 555556 570 Alexa Fluor ® 546 556 575 DY-547 557 574 DY-548 558 572 BODIPY ®558/568 558 569 DY-560 559 578 DY 549 560 575 DyLight ® 549 562 618 CF ™568 562 583 ATTO 565 563 592 BODIPY ® 564/570 565 571 CAL Fluor ® Red590 566 588 Lissamine rhodamine B 570 590 Rhodamine Red dye 570 590BODIPY ® 576/589 576 590 Alexa Fluor ® 568 578 603 X-rhodamine 580 605DY-590 580 599 BODIPY ® 581/591 584 592 CAL Fluor ® Red 610 587 608BODIPY ® TR 589 617 Alexa Fluor ® 594 590 617 ATTO 590 594 624 CF ™ 594594 614 CAL Fluor ® Red 615 595 615 Texas Red ® dye 595 615Naphthofluorescein 605 675 DY-682 609 709 DY-610 610 630 CAL Fluor ® Red635 611 631 ATTO 611x 611 681 Alexa Fluor ® 610 612 628 ATTO 610 615 634CF ™ 620R 617 639 ATTO 620 619 643 DY -615 621 641 BODIPY ® 630/650 625640 ATTO 633 629 657 CF ™ 633 630 650 Seta 632 632 641 Alexa Fluor ® 633632 647 Alexa Fluor ® 635 633 647 DY-634 635 658 Seta 633 637 647 DY-630636 657 DY-633 637 657 DY-632 637 657 DyLight ® 633 638 658 Seta 640 640656 CF ™ 640R 642 662 ATTO 647N 644 669 Quasar ® 670 644 670 ATTO 647645 669 DY-636 645 671 BODIPY ® 650/665 646 660 Seta 646 646 656 DY-635647 671 Square 635 647 666 Cy 5 649 650/670 Alexa Fluor ® 647 650 668CF ™ 647 650 665 Seta 650 651 671 Square 650 653 671 DY-647 653 672DY-648 653 674 DY-650 653 674 DyLight ® 649 654 673 DY-652 654 675DY-649 655 676 DY-651 656 678 Square 660 658 677 Seta 660 661 672 AlexaFluor ® 660 663 690 ATTO 655 663 684 Seta 665 667 683 Square 670 667 685Seta 670 667 686 DY-675 674 699 DY-677 673 694 DY-676 674 699 AlexaFluor ® 680 679 702 IRDye ® 700DX 680 687 ATTO 680 680 700 CF ™ 680R 680701 CF ™ 680 681 698 Square 685 683 703 DY-680 690 709 DY-681 691 708DyLight ® 680 692 712 Seta 690 693 714 ATTO 700 700 719 Alexa Fluor ®700 702 723 Seta 700 702 728 ATTO 725 725 752 ATTO 740 740 764 AlexaFluor ® 750 749 775 Seta 750 750 779 DyLight ® 750 752 778 CF ™ 750 755777 CF ™ 770 770 797 DyLight ® 800 777 794 IRDye ®800RS 770 786 IRDye ®800 CW 778 794 Alexa Fluor ® 790 782 805 CF ™ 790 784 806

Various embodiments of the multiplex panel may encompass a singlemultiplex dye system including at least five different dyes. The set ofamplification primers may be labeled with one of the at least fivedifferent dyes. The at least 5 different fluorescent dyes may bespectrally distinct. These at least five dyes may include any five ofthe above-listed dyes, or any other five dyes known in the art, or6-FAM™, VIC®, NED™, PET®, and LIZ™ dyes. Other embodiments may include asingle multiplex system comprising at least six different dyes. These atleast six dyes may include any six of the above-listed dyes, or anyother six dyes known in the art, 6-FAM™, VIC®, NED™, TAZ™, SID™, andLIZ™ dyes with the TAZ dye having a maximum emission at approximately600 nm, and the SID dye having a maximum emission at approximately 620nm (LIZ™ dye was used to label the size standards). The dyes may beenergy transfer dyes. An energy transfer dye has a donor dye whichabsorbs excitation energy and transfers energy to excite an acceptordye. The energy transfer dye may be configured to efficiently transferenergy from the donor dye to the acceptor dye. A multiplex energytransfer dye system having more than 1 dye may have the same dye as adonor dye for each of the energy transfer dye set member, while havingdifferent, spectrally distinct acceptor dyes for each member of the dyeset.

Allelic Ladder.

The allelic ladder serving as a reference standard and nucleic acid sizemarker for the amplified allele(s) from one or more STR markers of themultiplex panel may have one of many possible compositions. In someembodiments, the allelic ladder may include all known alleles for theSTR markers in the analysis. In other embodiments, the allelic laddermay not include all known alleles; additional alleles can be identifiedby size comparison with the existing allelic ladder components. In someembodiments, the allelic ladder can incorporate size standards for thealleles of different STRs, including a subset of all the STR markers ina multiplex analysis. Alternatively, the allelic ladder may include sizestandards for the alleles of all the STR markers in a multiplexanalysis. The allelic ladder for a multiplex analysis may be acombination of allelic ladders. In some embodiments, the allelic laddercan be DNA. In some embodiments the allelic ladder can includenon-naturally occurring nucleic acid analogs, which may further includenucleotide analogs. In some embodiments, the allelic ladder may includeboth naturally occurring nucleotides and nucleotide analogs. In someembodiments, the allelic ladder may include non-nucleotide moieties. Thedifferent individual size standards within an allelic ladder can, insome embodiments, be labeled with a detectable label, e.g., afluorophore. In some embodiments, the allelic ladder components arelabeled with the same fluorophore. In some embodiments, the allelicladder components are labeled with different fluorophores. The differentfluorophores may be spectrally distinct. The size standards can beselected to serve as reference for a specific pair (or pairs) ofoligonucleotide primers. For example if a first set of primers formarker X with a tetranucleotide repeat produces a 150 base pair ampliconcorresponding to allele 7, a corresponding first allelic laddercomponent will serve as a size standard for the 150 base amplicons. Thefirst set of primers for marker X may also produces a 154 base pairamplicon corresponding to allele 8 of marker X, and a correspondingsecond allelic ladder component will serve as a size standard for the154 base amplicons. Thus different size standards for different sizeamplicons of the same marker are contemplated. The size standard for agiven amplicon derived from a given allele may have a nucleic acid basesequence that is the same or different from the nucleic acid basesequence of the amplicon or allele from which the amplicon is derived.Following the construction of allelic ladders for individual loci, theladders may be electrophoresed at the same time as the amplificationproducts. Alternatively, the ladders may be electrophoresed separatelyafter a preselected number of analysis runs for comparison to theanalysis results. For allele analysis in electrophoresis systems, thesize standard can be selected to have the same electrophoretic mobilityas the amplicon of interest. Equivalent electrophoretic mobility of thesize standard and the amplicon or the allele from which the amplicon isderived may be achieved by including non-nucleotide moieties in eitherthe standard or the amplicon. Alternatively, in some embodiments, thesize standard can be selected to have a different electrophoreticmobility than the amplicon of interest. With an understanding of thepredicable nature of the difference in mobility between the sizestandard and the amplicon of interest, the identity of the amplicon maybe determined. For allele analysis in mass spectroscopy systems the sizestandard (weight/charge ratio, not electrophoretic mobility) can beselected so as to have the same signal as the amplicon of interest.Alternatively, in some embodiments, the size standard (weight/chargeratio, not electrophoretic mobility) can be selected to have differentseparation properties from the amplicon of interest. Similarly given anunderstanding of the predicable nature of weight/charge difference, theidentity of the amplicon may be determined. The individual size standardcomponents of the allelic ladder may be produced by expression,synthesis or semi-synthesis.

In one embodiment of allelic ladders for multiplex panels describedherein, the range (from the smallest allele to the largest allele) ofallelic size standards for each STR marker in the panel may be separatedby a two base pair difference from the preceding and following ranges ofallele size standards for the corresponding adjacent STR markers in themultiplex panel.

The range for each STR marker may also include a virtual bin at thesmallest end and the largest end of the range of alleles for future use.Additionally, software processing the electrophoretic separation resultsmay further be configured to assign virtual bins for microvarientalleles, for example, wherein only a partial repeat of the tandem repeathas occurred in the target nucleic acid.

One exemplary allelic ladder is shown in FIG. 15A. The allelic laddershown in FIG. 15A may be used to identify the alleles for the multiplexPanel 7. The first dye channel is shown in the top panel where the firstmarker is DYS576 with alleles 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24 and 25 detected in a region from about 70 bp to about135 bp; the second marker is DYS389I with alleles 9, 10, 11, 12, 13, 14,and 15 detected in a range from about 145 bp to about 175 bp; the thirdmarker is DYS635 with alleles 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, and 30 detected in a range from about 190 bp toabout 250 bp; the fourth marker is DYS389II with alleles 24, 25, 26, 27,28, 29, 30, 31, 32, 33, and 34 detected in a range from about 260 bp toabout 300 bp; and the fifth marker is DYS627 with alleles 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, and 25 detected in a range fromabout 320 bp to about 375 bp. The second dye channel is shown in thesecond panel from the top of FIG. 15A where the first marker is DYS460with alleles 7, 8, 9, 10, 11, 13, and 14 detected in a range from about75 bp to about 110 bp; the second marker is DYS458 with alleles 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 detected in a rangefrom about 115 bp to about 170 bp; the third marker is DYS19 withalleles 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 detected in a rangefrom about 180 bp to about 220 bp; the fourth marker is GATA-H4 withalleles 8, 9, 10, 11, 12, 13, 14, and 15 detected in a range from about220 bp to about 260 bp; the fifth marker is DYS448 with alleles 14, 15,16, 17, 18, 19, 20, 21, 22, 23, and 24 detected in a range from about275 bp to about 335 bp; and the sixth marker is DYS391 with alleles 5,6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 detected in a range from about345 bp to about 395 bp. The third dye channel is shown in the thirdpanel from the top of FIG. 15A where the first marker is DYS456 withalleles 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, and 24 detected in arange from about 75 bp to about 135 bp; the second marker is DYS390 withalleles 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28 and 29 detected in arange from about 140 bp to about 195 bp; the third marker is DYS438 withalleles 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 detected in a rangefrom about 200 bp to about 255 bp; the fourth marker is DYS392 withalleles 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, and 20detected in a range from about 265 bp to about 305 bp; and the fifthmarker is DYS518 with alleles 35, 36, 37, 38, 39, 40, 41, 42, 43 and 45detected in a range from about 340 bp to about 380 bp. The fourth dyechannel is shown in the fourth panel from the top of FIG. 15A, where thefirst marker is DYS570 with alleles 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, and 26 are detected in a range from about 95bp to about 165 bp; the second marker is DYS437 with alleles 10, 11, 12,13, 14, 15, 16, 17 and 18 detected in a range from about 175 bp to about205 bp; the third marker is DYS385ab with alleles 6, 7, 8, 9, 10, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28detected in a range from about 225 bp to about 305 bp; and the fourthmarker is DYS449 with alleles 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 37, 38, 40, and 41 detected in a range from about 325 bp toabout 400 bp. The fifth dye channel is shown in the fifth panel from thetop of FIG. 15A, where the first marker is DYS393 with alleles 7, 8, 9,11, 12, 13, 14, 15, 16, and 18 detected in a range from about 90 bp toabout 135 bp; the second marker is DYS439 with alleles 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 and 17 detected in a range from about 150 bp toabout 195 bp; the third marker is DYS481 with alleles 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32 detected in a rangefrom about 205 bp to about 255 bp; the fourth marker is DYF387S1ab withalleles 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44detected in a range from about 265 bp to about 320 bp, and the fifthmarker is DYS533 with alleles 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and17 detected in a range from about 340 bp to about 380 bp. The sixth dyechannel containing a size standard is not shown in FIG. 15A. In thisembodiment, virtual bins are provided at both the low by and the high bysides of the allele detection range for each marker.

In another embodiment, the allelic ladder is as above, with anadditional allele 10, for DYS456, to provide a range for DYS456 withalleles 10, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, and 24 detectedin a range from about 70 bp to about 135 bp. The additional range forDYS456 is shown specifically in FIG. 15B.

In yet another embodiment, the allelic ladder is as described for theallelic ladder of FIG. 15B, with the addition of allele 12 for DYS392.

In one aspect, the invention provides for an allelic ladder, where theallelic ladder includes at least one size standard for at least oneallele of at least 11 Y-STR markers, wherein the at least one sizestandard for the at least one allele of the at least 11 Y-STR markershas a base pair size of less than about 220 base pairs. The allelicladder may include a size standard for each of more than one allele ofthe at least 11 Y-STR markers, wherein each of the size standards has abase pair size of less than about 220 base pairs. The allelic ladder mayinclude a size standard for one or more alleles of the at least 11 Y-STRmarkers where the at least 11 Y-STR markers include DYS576, DYS389I,DYS460, DYS458, DYS19, DYS456, DYS390, DYS570, DYS437, DYS393, andDYS439. The allelic ladder may further include at least one sizestandard for at least one allele for at least five rapidly mutatingY-STR markers. The allelic ladder may include a size standard for eachof more than one allele of the at least five rapidly mutating Y-STRmarkers. The allelic ladder may include a size standard for each of morethan one allele of the at least five rapidly mutating Y-STR markerswhere the at least five rapidly mutating Y-STR markers includeDYF387S1ab, DYS449, DYS570, DYS576, and DYS627. The allelic ladder mayinclude a size standard for each of more than one allele of the at leastfive rapidly mutating Y-STR markers where the at least five rapidlymutating Y-STR marker further include DYS518. The allelic ladder mayinclude a size standard for each of more than one allele of the at least11 Y-STR markers where the at least 11 Y-STR markers include DYF387S1ab,DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393,DYS460, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS481,DYS518, DYS533, DYS570, DYS576, DYS627, DYS635, and Y-GATA-H4. Theallelic ladder may include a size standard for each of more than oneallele of the at least 11 Y-STR markers where the at least 11 Y-STRmarkers include DYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390,DYS391, DYS392, DYS393, and DYS460, DYS437, DYS438, DYS439, DYS448,DYS449, DYS456, DYS458, DYS481, DYS533, DYS570, DYS576, DYS627, DYS635,DYS643, and Y-GATA-H4. The allelic ladder may include at least one sizestandard for the at least one allele of the at least 11 Y-STR markers,where the at least one size standard is fluorescently labeled. Theallelic ladder may include a size standard for each of one or morealleles of the at least 11 Y-STR markers, where a plurality of sizestandards for the one or more alleles of each of the at least 11 Y-STRmarkers is labeled with one of five spectrally distinct fluorescentdyes. The allelic ladder may further include a size standard labeledwith a sixth dye, where the size standard provides a measure of basepair size.

The Sample.

In some embodiments, the sample encompassing the target nucleic acidbeing analyzed is from one or more of hair, feces, blood, tissue, urine,saliva, cheek cells, vaginal cells, skin, bone, tooth, buccal sample,amniotic fluid containing placental cells, and amniotic fluid containingfetal cells and semen. In some embodiments, the sample may originatefrom a crime scene, a sample associated with a crime scene, a sampletaken from a suspect, a reference sample or a sample taken from a humanunder consideration. In other embodiments, the sample may be anarcheological sample, a maternity sample, a paternity sample, a missingperson sample.

Methods.

In some embodiments, the present teachings relate to methods fordetecting and identifying alleles of a short tandem repeat (STR)sequence in a target nucleic acid. In some embodiments, the method fordetecting and identifying alleles of a STR sequence includes amplifyingat least one short tandem repeat sequence from a target nucleic acid bypolymerase chain reaction (PCR) using locus-specific oligonucleotideprimers. Following amplification, the amplification product's resultingamplified short tandem repeat sequence is compared with the amplifiedallelic ladder to call the allele based on matching the sample'samplification product to the allele standard found within the allelicladder. In some embodiments, the method for detecting and identifyingalleles of a short tandem repeat sequence uses PCR amplification of thetarget nucleic acid and employs oligonucleotide primer pairs. Themethods include workflows suitable for analysis of extracted DNA, whichmay be used for casework samples, as well as direct amplification, whichmay be used for single source samples.

Methods for analyzing nucleic acids are well known to one of skill inthe art as are methods for amplification by PCR. The analyses of the PCRamplification product, i.e., amplicon, includes, but is not limited to,detection, identification and in some instances, sequencing theamplification product, methods well established and known to one ofskill in the art. In some embodiments, the method for detecting andidentifying alleles of a short tandem repeat sequence involves comparingthe amplified short tandem repeat amplification sequence to thecorresponding allelic ladder by electrophoresis. Many electrophoresismethods for the separation of alleles are known to one of skill in theart and include, but are not limited to, denaturing and non-denaturinggel electrophoresis, capillary electrophoresis, and the like. Methodsfor sequencing the amplification product, e.g., Sanger sequencing arewell established and known to one of skill in the art.

In some embodiments of the present teachings, methods are providedwherein one or more samples are analyzed for the determination of STRalleles present in the sample. In some embodiments, the method includesisolating nucleic acid from the sample and PCR amplifying the nucleicacid to generate an amplification product.

The amplification product is then compared to an allelic ladder mixtureincluding one or more alleles per marker. Various embodiments of thepresent teachings relate to newly expanded groups of alleles of selectedY-STR loci. Embodiments of the claimed inventions include allelicladders for the detection of these novel alleles of the selected Y-STRloci.

Samples of genomic DNA can be prepared for use in the methods of thepresent teaching using any procedures for sample preparation that arecompatible with the subsequent amplification of DNA. Many suchprocedures are known by those skilled in the art. Some examples are DNApurification by phenol extraction (J. Sambrook et al. (1989), inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 9.14-9.19), andpartial purification by salt precipitation (S. Miller et al. (1988),Nucl. Acids Res. 16:1215) or chelex (PS Walsh et al. (1991),BioTechniques 10:506-513; CT Comey et al. (1994), J. Forensic Sci.39:1254) and the release of unpurified material using untreated blood(J. Burckhardt (1994), PCR Methods and Applications 3:239-243; RBEMcCabe (1991), PCR Methods and Applications 1:99-106; BY Nordvag (1992),BioTechniques 12:4 pp. 490-492).

Once a sample of genomic DNA is prepared, the target loci can beco-amplified in the multiplex amplification step of the presentteaching. Alternatively, the sample containing genomic DNA may bedirectly amplified from a substrate that the sample was collected upon,including but not limited to paper, fabric or fiber substrates.

Any of a number of different amplification methods can be used toamplify the loci, such as, for example, PCR (R K Saiki et al. (1985),Science 230: 1350-1354), transcription based amplification (D Y Kwoh andT J Kwoh (1990), American Biotechnology Laboratory, October, 1990) andstrand displacement amplification (SDA) (GT Walker et al. (1992), Proc.Natl. Acad. Sci., U.S.A. 89: 392-396). In some embodiments of thepresent teaching, multiplex amplification can be effected via PCR, inwhich the DNA sample is subjected to amplification using primer pairsspecific to each locus in the multiplex.

The chemical components of a standard PCR generally include a solvent,DNA polymerase, deoxyribonucleoside triphosphates (“dNTPs”),oligonucleotide primers, a divalent metal ion, and a DNA sample expectedto contain the target(s) for PCR amplification. Water can generally beused as the solvent for PCR, typically including a buffering agent andnon-buffering salts such as KCl. The buffering agent can be any bufferknown in the art, such as, but not limited to, Tris-HCl, and can bevaried by routine experimentation to optimize PCR results. Persons ofordinary skill in the art are readily able to determine optimalbuffering conditions. PCR buffers can be optimized depending on theparticular enzyme used for amplification.

Divalent metal ions can often be advantageous to allow the polymerase tofunction efficiently. For example, the magnesium ion is one which allowscertain DNA polymerases to function effectively. Typically MgCl₂ orMgSO₄ can be added to reaction buffers to supply the optimum magnesiumion concentration. The magnesium ion concentration required for optimalPCR amplification may depend on the specific set of primers and templateused. Thus, the amount of magnesium salt added to achieve optimalamplification is often determined empirically, and is a routine practicein the art. Generally, the concentration of magnesium ion for optimalPCR can vary between about 1 and about 10 mM. A typical range ofmagnesium ion concentration in PCR can be between about 1.0 and about4.0 mM, varying around a midpoint of about 2.5 mM. Alternatively, thedivalent ion manganese can be used, for example in the form of manganesedioxide (MnO₂), titrated to a concentration appropriate for optimalpolymerase activity, easily determined by one of skill in the art usingstandard laboratory procedures.

The dNTPs, which are the building blocks used in amplifying nucleic acidmolecules, can typically be supplied in standard PCR at a concentrationof, for example, about 40-200 μM each of deoxyadenosine triphosphate(“dATP”), deoxyguanosine triphosphate (“dGTP”), deoxycytidinetriphosphate (“dCTP”), and deoxythymidine triphosphate (“dTTP”). OtherdNTPs, such as deoxyuridine triphosphate (“dUTP”), dNTP analogs (e.g.,inosine), and conjugated dNTPs can also be used, and are encompassed bythe term “dNTPs” as used herein. While use of dNTPs at concentrations ofabout 40-200 μM each can be amenable to the methods of this teaching,concentrations of dNTPs higher than about 200 μM each could beadvantageous. Thus, in some embodiments of the methods of theseteachings, the concentration of each dNTP is generally at least about500 μM and can be up to about 2 mM. In some further embodiments, theconcentration of each dNTP may range from about 0.5 mM to about 1 mM.Specific dNTP concentrations used for any multiplex amplification canvary depending on multiplex conditions, and can be determinedempirically by one of skill in the art using standard laboratoryprocedures.

The enzyme that polymerizes the nucleotide triphosphates into theamplified products in PCR can be any DNA polymerase. The DNA polymerasecan be, for example, any heat-resistant polymerase known in the art.Examples of some polymerases that can be used in this teaching are DNApolymerases from organisms such as Thermus aquaticus, Thermusthermophilus, Thermococcus litoralis, Bacillus stearothermophilus,Thermotoga maritima and Pyrococcus sp. The enzyme can be acquired by anyof several possible methods; for example, isolated from the sourcebacteria, produced by recombinant DNA technology or purchased fromcommercial sources. Some examples of such commercially available DNApolymerases include AmpliTaq Gold® DNA polymerase; AmpliTaq Platinum®DNA polymerase; AmpliTaq® DNA Polymerase; AmpliTaq® DNA PolymeraseStoffel Fragment; rTth DNA Polymerase; and rTth DNA Polymerase, XL (allmanufactured by Applied Biosystems, Foster City, Calif.). Other examplesof suitable polymerases include Tne, Bst DNA polymerase large fragmentfrom Bacillus stearothermophilus, Vent and Vent Exo− from Thermococcuslitoralis, Tma from Thermotoga maritima, Deep Vent and Deep Vent Exo−and Pfu from Pyrococcus sp., and mutants, variants and derivatives ofthe foregoing.

Other known components of PCR can be used within the scope of thepresent teachings. Some examples of such components include sorbitol,detergents (e.g., Triton X-100, Nonidet P-40 (NP-40), Tween-20) andagents that disrupt mismatching of nucleotide pairs, such as, forexample, dimethylsulfoxide (DMSO), and tetramethylammonium chloride(TMAC), and uracil N-glycosylase or other agents which act to preventamplicon contamination of the PCR and/or unwanted generation of productduring incubation or preparation of the PCR, before the PCR procedurebegins.

PCR cycle temperatures, the number of cycles and their durations can bevaried to optimize a particular reaction, as a matter of routineexperimentation. Those of ordinary skill in the art will recognize thefollowing as guidance in determining the various parameters for PCR, andwill also recognize that variation of one or more conditions is withinthe scope of the present teachings. Temperatures and cycle times aredetermined for three stages in PCR: denaturation, annealing andextension. One round of denaturation, annealing and extension isreferred to as a “cycle.” Denaturation can generally be conducted at atemperature high enough to permit the strands of DNA to separate, yetnot so high as to destroy polymerase activity. Generally,thermoresistant polymerases can be used in the reaction, which do notdenature but retain some level of activity at elevated temperatures.However, heat-labile polymerases can be used if they are replenishedafter each denaturation step of the PCR. Typically, denaturation can beconducted above about 90° C. and below about 100° C. In someembodiments, denaturation can be conducted at a temperature of about94-95° C. Denaturation of DNA can generally be conducted for at leastabout 1 to about 30 seconds. In some embodiments, denaturation can beconducted for about 1 to about 15 seconds. In other embodiments,denaturation can be conducted for up to about 1 minute or more. Inaddition to the denaturation of DNA, for some polymerases, such asAmpliTaq Gold®, incubation at the denaturation temperature also canserve to activate the enzyme. Therefore, it can be advantageous to allowthe first denaturation step of the PCR to be longer than subsequentdenaturation steps when these polymerases are used.

During the annealing phase, oligonucleotide primers anneal to the targetDNA in their regions of complementarity and are substantially extendedby the DNA polymerase, once the latter has bound to the primer-templateduplex. In a conventional PCR, the annealing temperature can typicallybe at or below the melting point (T_(m)) of the least stableprimer-template duplex, where T_(m) can be estimated by any of severaltheoretical methods well known to practitioners of the art. For example,T_(m) can be determined by the formula:T _(m)=(4° C.×number of G and C bases)+(2° C.×number of A and T bases).

Typically, in standard PCR, the annealing temperature can be about 5-10°C. below the estimated T_(m) of the least stable primer-template duplex.The annealing time can be between about 30 seconds and about 2 minutes.The annealing phase is typically followed by an extension phase.Extension can be conducted for a sufficient amount of time to allow thepolymerase enzyme to complete primer extension into the appropriatelysized amplification products.

The number of cycles in the PCR (one cycle includes denaturation,annealing and extension) determines the extent of amplification and thesubsequent amount of amplification product. PCR results in anexponential amplification of DNA molecules. Thus, theoretically, aftereach cycle of PCR, there is twice the number of products that werepresent in the previous cycle, until PCR reagents are exhausted and aplateau is reached at which no further amplification products aregenerated. Typically, about 20-30 cycles of PCR may be performed toreach this plateau. More typically, about 25-30 cycles may be performed,although cycle number is not particularly limited. The number of cyclesused may depend on the nature of the input sample. In some cases, anextracted DNA sample may require 30 cycles, while a directly amplifiedDNA sample may only require 26 cycles. One of skill may adjust bothcycle numbers and specific details of temperature and time intervals inorder to optimize the reaction conditions.

For some embodiments, it can be advantageous to incubate the reactionsat a certain temperature following the last phase of the last cycle ofPCR. In some embodiments, a prolonged extension phase can be selected.In other embodiments, an incubation at a low temperature (e.g., about 4°C.) can be selected.

Various methods can be used to evaluate the products of the amplifiedalleles in the mixture of amplification products obtained from themultiplex reaction including, for example, detection of fluorescentlabeled products, detection of ions released during each extensionreaction (ion semiconductor sequencing), detection of pyrophosphaterelease during each extension reaction, detection of radioisotopelabeled products, silver staining of the amplification products, or theuse of DNA intercalator dyes such as ethidium bromide (EtBr) and SYBRgreen cyanine dye to visualize double-stranded amplification products.Fluorescent labels suitable for attachment to primers for use in thepresent teachings are numerous, commercially available, and well-knownin the art. With fluorescent analysis, at least one fluorescent labeledprimer can be used for the amplification of each locus. Fluorescentdetection may be desirable over radioactive methods of labeling andproduct detection, for example, because fluorescent detection does notrequire the use of radioactive materials, and thus avoids the regulatoryand safety problems that accompany the use of radioactive materials.Fluorescent detection with labeled primers may also be selected overother non-radioactive methods of detection, such as silver staining andDNA intercalators, because fluorescent methods of detection generallyreveal fewer amplification artifacts than do silver staining and DNAintercalators. This is due in part to the fact that only the amplifiedstrands of DNA with labels attached thereto are detected in fluorescentdetection, whereas both strands of every amplified product are stainedand detected using the silver staining and intercalator methods ofdetection, which result in visualization of many non-specificamplification artifacts. Additionally, there are potential health risksassociated with the use of EtBr and SYBR. EtBr is a known mutagen; SYBR,although less of a mutagen than EtBr, is generally suspended in DMSO,which can rapidly pass through skin.

Fluorescence detection may also be useful in sequencing by synthesismethods, where each nucleotide extension reaction releases a fluorescentsignal that is differentiable for each of the four natural nucleotides.Nothing in this disclosure would limit one of skill from using suchdetection methods with the primers and of the multiplex panels of theinvention, and methods described herein.

Detection of the amplified alleles is not limited, however, tofluorescence detection and may be conveniently detected viapyrosequencing or ionic semiconductor sequencing techniques. Theselection of core loci in combination with additional loci selected forgene diversity and/or high mutational rate makes the multiplex assaysdescribed herein equally useful for those detection methodologies.

Improved Capabilities of the Panels in the Methods of the Invention.

The Panel 6 and Panel 7 multiplex experimental results demonstrate theincreased sensitivity, robustness, and discriminatory power of themultiplex assay panels designed and performed as described here. Asshown in Examples 1 and 2, both Panel 6 and Panel 7 provide higherproportions of complete Y-STR profiles even at very low levels of inputDNA. Examples 3, 4, and 5 show that the primer sets of Panel 6 and Panel7 permit higher proportion of complete Y-STR profiles for decreasingamounts of male DNA in the presence of increasing amounts of female DNA,relative to a commercially available panel. Thus the primers of Panel 6and Panel 7 offer higher specificity for male DNA compared to theY®filer panel. Examples 6 and 7 demonstrate that the primer sets ofPanel 6 and Panel 7 provide improved intracolor peak balance overall andalso in the presence of increasing amounts of female DNA in the inputsample. Intracolor balance is defined below, but represents a measure ofrobust and equivalent amplification across all alleles for which theprimers are labeled with the same fluorescent dye. This measure isanother mark of the specificity of the primers with respect to thedesired target male DNA vs hybridization or association with inhibitorsor with female DNA. Examples 8, 9, and 10 demonstrate that the primersets and chemistry used for Panel 6 and 7 provide more significantlyrobust amplification of target male DNA in the presence of varyingconcentrations of PCR inhibitors such as humic acid or hematin, comparedto the amplification seen with the Y®filer panel. These results areparticularly important for forensic or crime scene samples. Example 11shows the improved results possible using the more robust intracolorbalance characteristics of Panel 6 or 7, in order to identify minor malecontributor to a mixed male input DNA sample. As shown in Examples 13and 15, Panel 7 demonstrates increased ability to differentiate betweena father and son, compared to other commercially available multiplexY-STR panels. This improved differentiation (alternatively, ability todiscriminate between a father and son) affords a greater possibility ofidentifying a male individual specifically than previously possible.This improved discrimination also affords the ability to more positivelyexclude a male individual from consideration as a potential lead incriminal, forensic or other evidentiary scenarios. Example 14demonstrates the increased ability to resolve haplotypes using Panel 7multiplex relative to the use of Y®filer, including groups havinggreater ethnic variety, i.e., in more groups of non-European lineage.Therefore, the multiplex panels of primers, and the methods of usethereof, offer surprisingly robust, sensitive, and specific Y-STRprofile results. These improvements are badly needed in the field offorensics, human identification and justice.

A method is provided to amplify alleles of Y-STR markers of a human maleincluding the steps of: contacting a sample suspected to contain a DNAsample of a human male with a set of amplification primers includingprimers for the amplification of the alleles of at least 11 Y-STRmarkers; and amplifying the sample thereby forming a plurality of setsof amplicons of the at least 11 Y-STR markers where each set of theamplicons has a base pair size less than about 220 base pairs. Themethod may further include the step of detecting each set of ampliconswhereby the alleles of the at least 11 Y-STR markers are identified. Insome embodiments, the detecting step is a fluorescence detection step.In some embodiments, the detecting step is performed by separating theplurality of sets of amplicons using a mobility dependent analysis,where the plurality of sets of amplicons is fluorescently labeled. Inother embodiments, the detecting step does not detect fluorescence. Inembodiments, when the detecting step does not detect fluorescence, thedetecting step may include ion semiconductor detection, pyrophosphaterelease detection, or mass spectrometry detection. In variousembodiments of the method, the set of amplification primers may furtherinclude primers for the amplification of at least 5 Y-STR markers wherethe primers may be configured to provide each set of amplicons of the atleast 5 Y-STR markers having a base pair size greater than about 220base pairs. In various embodiments of the method, when the set ofamplification primers amplifies more than 11 Y-STR markers, then the setof primers may be configured to provide all of the sets of amplicons ofthe more than 11 Y-STR markers having a base pair size less than about400 base pairs. In various embodiments of the method, when the set ofamplification primers amplifies more than 11 Y-STR markers, then the setof primers may be configured to provide all of the sets of amplicons ofthe more than 11 Y-STR markers having a base pair size less than about410 base pairs. In various embodiments of the method, when the set ofamplification primers amplifies more than 11 Y-STR markers, then the setof primers may be configured to provide all of the sets of amplicons ofthe more than 11 Y-STR markers having a base pair size less than about420 base pairs. In some embodiments of the method, when theamplification primer set amplifies more than 11 Y-STR markers, then theprimer set may include primers for the amplification of 25 Y-STRmarkers. In some embodiments, the primer set for the amplification of 25Y-STR markers, includes at least two double copy markers. In someembodiments, the set of amplification primers may be labeled with one ofat least 5 fluorescent dyes. In some other embodiments, each set of theamplicons of the at least 11 Y-STR markers may be labeled with one of atleast 5 fluorescent dyes. In various embodiments of the method, the atleast 5 fluorescent dyes used to label the primers and/or the ampliconsmay be configured to be spectrally distinct. The set of amplificationprimers used in the method may further include at least oneamplification primer that includes a mobility modifier. In someembodiments of the method, the at least one set of amplicons may includea mobility modifier. In various embodiments of the methods, the set ofamplification primers amplifying at least 11 Y-STR markers, may amplifyDYS576, DYS389I, DYS460, DYS458, DYS19, DYS456, DYS390, DYS570, DYS437,DYS393, and DYS439. In other embodiments, the set of amplificationprimers amplifying the at least 11 Y-STR markers, may amplify at least 5Y-STR markers which are rapidly mutating loci. In some embodiments, theat least 5 rapidly mutating Y-STR markers may include DYF387S1ab,DYS449, DYS570, DYS576, and DYS627. In other embodiments, the at least 5rapidly mutating Y-STR markers may further include DYS518. In someembodiments of the method, the set of primers for the amplification ofat least 11 Y-STR markers may be a set of primers for the amplificationof DYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391,DYS392, DYS393, DYS460, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456,DYS458, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627, DYS635, andY-GATA-H4. In other embodiments, the set of primers for theamplification of at least 11 Y-STR markers may be a set of primers forthe amplification of DYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II,DYS390, DYS391, DYS392, DYS393, DYS460, DYS437, DYS438, DYS439, DYS448,DYS449, DYS456, DYS458, DYS481, DYS533, DYS570, DYS576, DYS627, DYS635,DYS643, and Y-GATA-H4. In some embodiments, the method includes a set ofamplification primers for the amplification of the alleles of 27 Y-STRmarkers.

In another aspect, a method is provided to amplify alleles of Y-STRmarkers of a human male including the steps of: contacting a samplesuspected to contain a DNA sample of a human male with a set ofamplification primers including primers for the amplification of thealleles of at least 22 Y-STR markers, wherein at least 5 of the Y-STRmarkers are rapidly mutating loci; and amplifying the sample therebyforming a plurality of sets of amplicons of the at least 22Y-STRmarkers. In some embodiments of the method, a set of amplificationprimers of the alleles of at least 23 Y-STR markers are provided,wherein at least 5 of the Y-STR markers are rapidly mutating loci. Inyet other embodiments, a set of amplification primers of the alleles of27 Y-STR markers are provided, wherein at least 5 of the Y-STR markersare rapidly mutating loci. In some embodiments, the 27 Y-STR markersinclude 2 Y-STR markers having double copy markers contributing to thetotal number of Y-STR markers. In some embodiments, each set of theamplicons of at least 11 of the at least 22 Y-STR markers has a basepair size less than about 220 base pairs. In other embodiments, each setof the amplicons of at least 11 of at least 23 Y-STR markers has a basepair size less than about 220 base pairs. In yet other embodiments, eachset of the amplicons of at least 11 of 27 Y-STR markers has a base pairsize less than about 220 base pairs. In various embodiments of themethod, a set of amplification primers including primers for theamplification of the alleles of at least 22 Y-STR markers are provided,wherein at least 6 of the Y-STR markers are rapidly mutating loci. Invarious embodiments of the method, a set of amplification primersincluding primers for the amplification of the alleles of at least 22Y-STR markers are provided, wherein at least 7 of the Y-STR markers arerapidly mutating loci. The method may further include the step ofdetecting each set of amplicons whereby the alleles of at least 22 Y-STRmarkers are identified. In some embodiments, the alleles of at least 23Y-STR markers are identified. In yet other embodiments, the alleles of27 Y-STR markers are identified. In some embodiments, the detecting stepis a fluorescence detection step. In some embodiments, the detectingstep is performed by separating the plurality of sets of amplicons usinga mobility dependent analysis, where the plurality of sets of ampliconsis fluorescently labeled. In other embodiments, the detecting step doesnot detect fluorescence. In embodiments, when detecting steps do notdetect fluorescence, the detecting step may include ion semiconductordetection, pyrophosphate release detection, or mass spectrometrydetection. In some embodiments, the at least 11 Y-STR markers havingamplicons having a base pair size of less than about 220 base pairs areDYS576, DYS389I, DYS460, DYS458, DYS19, DYS456, DYS390, DYS570, DYS437,DYS393, and DYS439. In some the embodiments, the at least 5 rapidlymutating Y-STR markers are selected from the group consisting ofDYF387S1ab, DYS449, DYS518, DYS570, DYS576, and DYS627. In otherembodiments, the at least 5 rapidly mutating Y-STR markers are 6 rapidlymutating Y-STR markers.

A method of male individual identification, including the steps of:contacting a sample containing a nucleic acid of a human male with a setof amplification primers including primers for the amplification of thealleles of at least 11 Y-STR markers; and amplifying the sample therebyforming a plurality of sets of amplicons of the at least 11 Y-STRmarkers where each set of the amplicons has a base pair size less thanabout 220 base pairs; and detecting each set of amplicons whereby thealleles of the male individual are identified. In various embodiments ofthe methods, the set of amplification primers amplifying at least 11Y-STR markers, may amplify DYS576, DYS389I, DYS460, DYS458, DYS19,DYS456, DYS390, DYS570, DYS437, DYS393, and DYS439. In otherembodiments, the step of amplifying the at least 11 Y-STR markers, mayinclude amplifying at least 5 Y-STR markers which are rapidly mutatingloci. In some embodiments, the at least 5 rapidly mutating Y-STR markersmay include DYF387S1ab, DYS449, DYS570, DYS576, and DYS627. In otherembodiments, the at least 5 rapidly mutating Y-STR markers may furtherinclude DYS518. In some embodiments of the method, the set of primersfor the amplification of at least 11 Y-STR markers may be a set ofprimers for the amplification of DYF387S1ab, DYS19, DYS385ab, DYS389I,DYS389II, DYS390, DYS391, DYS392, DYS393, DYS460, DYS437, DYS438,DYS439, DYS448, DYS449, DYS456, DYS458, DYS481, DYS518, DYS533, DYS570,DYS576, DYS627, DYS635, and Y-GATA-H4. In other embodiments, the set ofprimers for the amplification of at least 11 Y-STR markers may be a setof primers for the amplification of DYF387S1ab, DYS19, DYS385ab,DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS460, DYS437,DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS481, DYS533, DYS570,DYS576, DYS627, DYS635, DYS643, and Y-GATA-H4. In some embodiments, themethod includes a set of amplification primers for the amplification ofthe alleles of more than 11Y-STR markers. In other embodiments, theplurality of sets of amplicons of the more than 11 Y-STR markers wherethe plurality of sets of the amplicons has a base pair size less thanabout 410 base pairs. In some embodiments, the detecting step is afluorescence detection step. In some embodiments, the method furtherincludes the step of comparing the alleles identified for a first maleindividual to the alleles identified for a second male individual,whereby the first male individual is differentiable from the second maleindividual. In some embodiments, the first male individual has a similarpaternal genetic lineage as the second male individual.

Kits.

In some embodiments, the present teachings are directed to kits foranalyzing a short tandem repeat sequence from a nucleic acid sample thatutilize the methods described above. In some embodiments, a kit foranalyzing a short tandem repeat sequence in a nucleic acid sampleincludes at least one receptacle containing a set of primers configuredto hybridize to Y-STR markers. The kit may include primers for theamplification of at least 11 Y-STR markers where the primers areconfigured to provide each set of amplicons of the at least 11 Y-STRmarkers having a base pair size less than about 220 base pairs; andoptionally, a size standard. In some embodiments, the kit may furtherinclude primers for the amplification of at least 5 Y-STR markers wherethe primers are configured to provide each set of amplicons of the atleast 5 Y-STR markers having a base pair size greater than about 220base pairs. The kit may include an amplification primer set for 25 Y-STRmarkers. In various embodiments, when the set of amplification primersamplify more than 11 Y-STR markers, then the set of amplificationprimers may be configured to provide all of the sets of amplicons of themore than 11 Y-STR markers having a base pair size less than about 410base pairs. In various embodiments, when the set of amplificationprimers amplify more than 11 Y-STR markers, then the set ofamplification primers may be configured to provide all of the sets ofamplicons of the more than 11 Y-STR markers having a base pair size lessthan about 420 base pairs. In some embodiments, the kit may include aset of amplification primers labeled with one of at least 5 fluorescentdyes. The at least 5 fluorescent dyes used to label the primers of thekit may be configured to be spectrally distinct. The kit may furtherinclude at least one amplification primer that includes a mobilitymodifier. In some embodiments, the kit including a set of amplificationprimers amplifying at least 11 Y-STR markers, may amplify DYS576,DYS389I, DYS460, DYS458, DYS19, DYS456, DYS390, DYS570, DYS437, DYS393,and DYS439. In other embodiments, the kit including a set ofamplification primers amplifying the at least 11 Y-STR markers, wherethe primers are configured to provide each set of amplicons of the atleast 11 Y-STR markers having a base pair size less than about 220 basepairs, may amplify at least 5 Y-STR markers which are rapidly mutatingloci. In some embodiments, the at least 5 rapidly mutating Y-STR markersmay include DYF387S1ab, DYS449, DYS570, DYS576, and DYS627. In otherembodiments, the at least 5 rapidly mutating Y-STR markers may furtherinclude DYS518. In some embodiments, the kit including a set of primersfor the amplification of at least 11 Y-STR markers may be a set ofprimers for the amplification of DYF387S1ab, DYS19, DYS385ab, DYS389I,DYS389II, DYS390, DYS391, DYS392, DYS393, DYS460, DYS437, DYS438,DYS439, DYS448, DYS449, DYS456, DYS458, DYS481, DYS518, DYS533, DYS570,DYS576, DYS627, DYS635, and Y-GATA-H4. In other embodiments, the kitincluding a set of primers for the amplification of at least 11 Y-STRmarkers may be a set of primers for the amplification of DYF387S1ab,DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393,DYS460, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS481,DYS533, DYS570, DYS576, DYS627, DYS635, DYS643, and Y-GATA-H4. In someembodiments, the kit includes a size standard. In various embodiments,the kit further includes an allelic ladder.

In another aspect, a kit for co-amplifying a set of loci of at least oneDNA sample may be provided, including a set of amplification primers forthe amplification of at least 22 Y-STR markers where at least 5 of theY-STR markers are rapidly mutating loci; and optionally, a sizestandard. In some embodiments, the size standard is an allelic ladder.In some embodiments, the at least 5 rapidly mutating Y-STR markers ofthe kit include DYF387S1ab, DYS449, DYS570, DYS576, and DYS627. In someembodiments, the at least 5 rapidly mutating Y-STR markers includeDYS518. In some embodiments, the set of amplification primers is labeledwith one of at least 5 fluorescent dyes. The set of amplificationprimers of the kit for the amplification of at least 22 Y-STR markersmay be configured to provide at least one set of amplicons of the Y-STRmarkers where the at least one set of amplicons includes a mobilitymodifier. In some embodiments, the at least 22 Y-STR markers may includeDYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392,DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS570,DYS576, DYS627, DYS635, and Y-GATA-H4. In other embodiments, the atleast 22 Y-STR markers may include DYF387S1ab, DYS19, DYS385ab, DYS389I,DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439,DYS448, DYS449, DYS456, DYS458, DYS518, DYS570, DYS576, DYS627, DYS635,and Y-GATA-H4.

In some embodiments, a sufficient quantity of enzyme for amplification,amplification buffer to facilitate the amplification, a divalent cationsolution to facilitate enzyme activity, dNTPs for strand extensionduring amplification, loading solution for preparation of the amplifiedmaterial for electrophoresis, genomic DNA as a template control, a sizemarker to insure that materials migrate as anticipated in the separationmedium, and a protocol and manual to educate the user and limit error inuse may be included in the kits of the invention in any combination orselection. The amounts of the various reagents in the kits also can bevaried depending upon a number of factors, such as the optimumsensitivity of the process. It is within the scope of these teachings toprovide test kits for use in manual applications or test kits for usewith automated sample preparation, reaction set-up, detectors oranalyzers. Those in the art understand that the detection techniquesemployed are generally not limiting. Rather, a wide variety of detectionmeans are within the scope of the disclosed methods and kits, providedthat they allow the presence or absence of an amplicon to be determined.

While the principles of this invention have been described in connectionwith specific embodiments, it should be understood clearly that thesedescriptions are made only by way of example and are not intended tolimit the scope of the invention. What has been disclosed herein hasbeen provided for the purposes of illustration and description. It isnot intended to be exhaustive or to limit what is disclosed to theprecise forms described. Many modifications and variations will beapparent to the practitioner skilled in the art. What is disclosed waschosen and described in order to best explain the principles andpractical application of the disclosed embodiments of the art described,thereby enabling others skilled in the art to understand the variousembodiments and various modifications that are suited to the particularuse contemplated. It is intended that the scope of what is disclosed bedefined by the following claims and their equivalence.

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

EXAMPLES

Those having ordinary skill in the art will understand that manymodifications, alternatives, and equivalents are possible. All suchmodifications, alternatives, and equivalents are intended to beencompassed herein.

The following procedures are representative of reagents and proceduresthat can be employed for the isolation and amplification of a targetnucleic acid in a sample and the detection, identification and analysisof short tandem repeat sequences on the Y chromosome.

PCR Assay Set-Up and Reaction Conditions.

The primer sets of Panels 1-7 are prepared as described above anddye-labeled and unlabeled primers in buffer (low EDTA buffer containing10 mM Tris-HCL, pH 8.0, and 0.1 mM EDTA, pH 8.0) are amplified in PCRreactions as follows:

1 ng input DNA, typically, is used.

Add 10 μL Master Mix which includes polymerase enzyme.

Add 5 μL primer set.

Mix thoroughly by vortexing at medium speed for 10 sec. Centrifugebriefly to remove any liquid from the cap of the tube.

Add 15 μL of the PCR reaction mixture to each reaction well or tube.

Centrifuge plate or tubes at 3000 rpm for about 30 sec to remove anybubbles prior to amplification. Amplify in GeneAmp® PCR System 9700thermocycler or Venti® 96-well Thermal Cycler, for 26-30 cycles, usingthe following sequence: hold at 95° C. for 1 min; denature at 94° C. for4 sec; anneal/extend at 61° C. for 1 min; final extension at 60° C. for10 min; and hold at 4° C. for storage or until analysis. Samples runusing Yfiler® primer sets are prepared and amplified according to theAmpFISTR® Yfiler® PCR Amplification Kit User's Guide (Part no. 4359513).

Capillary Electrophoresis Sample Preparation and Detection.

The amplified samples are analyzed by methods that resolve amplificationproduct size and/or sequence differences as would be known to one ofskill in the art. For example, capillary electrophoresis can be usedfollowing the instrument manufactures directions. Briefly, 0.5 μLGeneScan™-600 LIZ™ Size Standard and 9.5 μL of Hi-Di™ Formamide aremixed for each sample to be analyzed. 10.0 μL of theFormamide/GeneScan-600 LIZ solution is dispensed into each well of aMicroAmp® Optical 96-well reaction plate to which a 1.0 μL aliquot ofthe PCR amplified sample or allelic ladder is added and the plate iscovered. The plate is briefly centrifuged to mix the contents andcollect them at the bottom of the plate. The plate is heated at 95° C.for 3 min to heat-denature the samples and then quenched immediately byplacing on ice for 3 min.

Capillary Electrophoresis Methods and Analysis.

Capillary electrophoresis (CE) was performed on current AppliedBiosystems instruments: the Applied Biosystems 3500xl Genetic Analyzerusing the specified J6 variable binning module as described in theinstrument's User's Guide. The 3500xl Genetic Analyzer's parameterswere: sample injection for 24 sec at 1.2 kV and electrophoresis at 15 kVfor 1550 sec in Performance Optimized Polymer (POP-4™ polymer) with arun temperature of 60° C. as indicated in the HID36_POP4xl_G5_NT3200protocol. Variations in instrument parameters, e.g. injectionconditions, were different on other CE instruments such as the 3500,3130xl, or 3130 Genetic Analyzers.) The data were collected usingversions of the Applied Biosystems Data Collection Software specific tothe different instruments, such as v.3.0 for the 3130xl and 3500 DataCollection Software v.1.0, was analyzed using GeneMapper ID-X v1.2.

Following instrument set-up according to the manufacturer's directionseach sample is injected and analyzed by appropriate software, e.g.,GeneMapper® ID-X v1.2 software with the standard analysis settings. Apeak amplitude of 175 RFU (relative fluorescence units) was used as thepeak detection threshold.

Example 1

Sensitivity study. The effect of decreasing target male DNA is compared,using the Panel 6 multiplex or the Yfiler® multiplex, as shown in FIG.16. The DNA input is varied from 1 ng, 500 pg, 250 pg, 63 pg, and 31 pg,with N=6. The Y filer multiplex begins to suffer allelic dropout at 63pg. While there are some losses using the Panel 6 multiplex, the overallrobustness of identification is higher because Panel 6 has 27 allelesvs. Yfiler®'s 17. More reliable identification is possible using Panel 6multiplex at lower concentrations of target male DNA.

Example 2

Sensitivity study. The effect of decreasing target male DNA is compared,using the Panel 7 multiplex or the Yfiler® multiplex, as shown in FIG.17. The data shown is collected from four different test sites, eachperforming N-4 replications. The DNA input is varied from 1 ng, 500 pg,250 pg, 62.5 pg, and 32.25 pg. The average number of alleles identifiedat each concentration for each multiplex is indicated by thecrosshatched circle symbol. The Y filer multiplex begins to sufferallelic dropout at 63 pg. While there are some losses using the Panel 7multiplex, the overall robustness of identification is higher becausePanel 7 has 27 alleles vs. Yfiler®'s 17. More reliable identification ispossible using Panel 7 multiplex at lower concentrations of target maleDNA.

Example 3

The effect of using male/female DNA mixtures as input DNA is compared,using the Panel 6 multiplex or the Yfiler® multiplex, as shown in FIG.18. As male DNA input is decreased (1 ng, 500 pg, 250 pg, 125 pg, 63 pg,to 31 pg), in the presence of constant female DNA input of 500 ng, withN=6. The Panel 6 multiplex recovers a higher percentage of alleles thanthe Yfiler® multiplex recovers. Even at 31 pg of male target DNA, over70% of alleles of the Panel 6 multiplex are identified.

Example 4

The effect of using male/female DNA mixtures as input DNA is compared,using the Panel 7 multiplex or the Yfiler® multiplex, as shown in FIG.19. The data shows results from a total of four test sites, where maleDNA input is decreased from 1 ng to 0.5 ng (M007) in the presence ofconstant female DNA input of 1 ug (F9947), with N=3. The left panelshows the results for the 1 ng M007/1 ug F9947 and the right panel showsthe results for 0.5 ng/1 ug F9947 for both Panel 7 and Yfiler®multiplexes, with averages for each multiples shown by a crosshatchedcircle. The Panel 7 multiplex recovers a higher percentage of allelesthan the Yfiler® multiplex recovers. Even at a 1:2000 ratio ofmale:female, all alleles of the Panel 7 multiplex are identified.

Example 5

The effect of using male/female DNA mixtures as input DNA is compared,using the Panel 6 multiplex or the Yfiler® multiplex, as shown in FIG.20. As male target DNA input is held constant at 500 pg, female DNA isincreased from 500 ng, to 1 μg, to 2 μg, with N=6. At the highestconcentration of female DNA, over 90% of alleles in the Panel 6multiplex are identified whereas less than 60% of alleles are identifiedin the Yfiler® multiplex.

Example 6

Intracolor balance in male/female mixtures is compared, using the Panel6 multiplex or Yfiler® multiplex, as shown in FIG. 21. Male target DNAinput is held constant (500 pg) and female DNA concentration is variedfrom 0.0 ng to 500 ng, for each of the five dye channels containingalleles of the multiplex, with N=6. Intracolor peak balance (ICB) iscalculated by dividing the lowest peak height by the highest peak heightwithin a color, i.e., all the markers labeled with the same fluorophoreand detectable in the same wavelength region. An ICB value of 1 wouldmean that all the alleles labeled with the same fluorophore havecompletely uniform peak heights, and any improvement of the ICB ratiotowards a value of 1 may offer higher accuracy in identifying thepresence of a particular allele. As shown in FIG. 21, Panel 6 multiplexis significantly less affected than the Yfiler® multiplex by thepresence of female DNA, having ICB values consistently greater thanabout 0.50. Additionally, all five dye channels behave more similarly toeach other than that of the Yfiler® multiplex.

Example 7

Intracolor balance in male/female mixtures is compared, using the Panel7 multiplex or Yfiler® multiplex, as shown in FIG. 22. The two panels onthe left side of the figure represents, in the first column: Male targetDNA M007 at 1 ng and female DNA F9947 at 1 ug (1:1000 M:F) and in thesecond column: Male target DNA M007 at 500 pg and female DNA F9947 at 1ug (1:2000 M:F), for each of the five dye channels (●, ▪, ⋄, ▴, and

) containing alleles of the Panel 7 multiplex, from a total of 4 testsites, each with N=3. The two panels on the right side of the figurerepresents, in the first column: Male target DNA M007 at 1 ng and femaleDNA F9947 at 1 ug (1:1000 M:F) and in column 2: Male target DNA M007 at500 pg and female DNA F9947 at 1 ug (1:2000 M:F), for each of the fourdye channels (●, ▪, ⋄, and ▴) containing alleles of the Yfiler®multiplex, from a total of 4 test sites, each with N=3. Intracolor peakbalance (ICB) is calculated and defined as described in Example 6. Asshown in FIG. 22, Panel 6 multiplex is significantly less affected thanthe Yfiler® multiplex by the presence of female DNA.

Example 8

Recovery of alleles in the presence of humic acid, an inhibitor of PCRare compared, using the Panel 6 multiplex or the Yfiler® multiplex, asshown in FIG. 23. Male target DNA input is held constant at 500 pg, andthe concentration of humic acid is increased from 0.0 ng/μl, 12.5 ng/μl,25 ng/μl, and 100 ng/μl, using N=6. As the concentration of humic acidis raised to 12.5 ng/μl or higher, the recovery of alleles using theYfiler® multiplex is severely inhibited. The use of the Panel 6multiplex permits recovery of nearly 80% of the alleles even at thehighest concentration of humic acid in the amplification reaction.

Example 9

Recovery of alleles in the presence of hematin, an inhibitor of PCR arecompared, using the Panel 6 multiplex or the Yfiler® multiplex, as shownin FIG. 24. Male target DNA input is held constant at 500 pg, and theconcentration of hematin is increased from 0.0 μM, 15 μM, 30 μM, and 120μM, using N=6. As the concentration of hematin is raised to 30 μM orhigher, the recovery of alleles using the Yfiler® multiplex is blockedcompletely. The use of the Panel 6 multiplex permits recovery of all ofthe alleles even at the highest concentration of hematin in theamplification reaction.

Example 10

Recovery of alleles in the presence of hematin, an inhibitor of PCR, orin the presence of humic acid, are compared using the Panel 7 multiplexor the Yfiler® multiplex, as shown in FIG. 25. Male target DNA input isheld constant at 1 ng, and hematin is present at 20 μM (column 1);hematin is present at 180 μM (column 2); humic acid is present at 10ng/μl (column 3); or humic acid is present at 80 ng/μl (column 4) datafrom a total of four test sites each using N=4. As the concentration ofhematin is raised to 180 μM or higher, the recovery of alleles using theYfiler® multiplex is blocked completely. The use of the Panel 7multiplex permits recovery of all of the alleles even at the highestconcentration of hematin in the amplification reaction. At humic acidconcentrations of 80 ng/ul, recovery of alleles using the Yfiler®multiplex is blocked completely, while the use of Panel 7 multiplexpermits complete recovery. FIG. 26 shows the intracolor balance for eachof the five dye channels (circle, square, diamond, ▴, and

) of this data. As can be seen for each of the two differingconcentrations of hematin and humic acid, respectively, use of the Panel7 multiplex provides more consistent results across all dye channels.

Example 11

The ability to identify a minor male contributor in a mixture of 2 malecontributor target DNA samples is shown in FIG. 27. An 8:1 ratio of 437pg:63 pg major:minor contributor is shown in the segment of theelectropherogram shown. The upper trace shows the mixture, with arrowspointing to the alleles belonging to the minor male contributor. This iscompared to the minor male contributor profile shown in the lower panelof FIG. 27, which was obtained without the presence of any other maleDNA sample. The improved intracolor balance provided by the primers usedin Panel 6 or Panel 7 yields improved uniformity of peak heights acrossan allele range, thus allowing improved identification of minorcontributor alleles.

Example 12

Direct Amplification. The primers directed to multiplex Panel 7 are usedto directly amplify unprocessed biological samples. The sample types areblood collected on FTA paper (n=3); buccal sample on FTA paper (n=3);and swab lysate (3 different swab materials, all aged from about 6months to about 1 year after collection). The primers used for loci inPanel 7 that are also present in Panel 6 have identical sequences, andtherefore have equivalent performance. The Mastermix used foramplification is adapted for universal application. FIG. 28 shows thecomparison of intracolor balance for each of the replicates of the threedifferent sample/substrate types. The results show that the minimallyrequired signal threshold of greater than 0.40 is achieved across allthree sample/substrate types for direct amplification. FIG. 29 shows anelectropherogram of the resulting Y-STR profile of one of the blood onFTA samples. For each color panel, full scale is 10,000 Rfus. It isshown that complete and unambiguous profiles are obtained with thedirect amplification method.

Example 13

Father-Son Study. Fifty three father-son pairs are studied using themultiplex of Panel 7, Yfiler®, and another commercially available kit,Kit B. In Table 4, the results are presented. A number of mutations areidentified, for which the total is shown in column 2, and the locationof the mutation is shown in column 1 Empty cells in the table indicatewhere a mutation was not identified because that marker is not part ofthe panel. The total number of mutations/Kit represent the number ofmutations identified overall for each kit. As can be seen, the Yfiler®multiplex can identify 4 mutations; Kit B can identify 5 mutations, andPanel 7 identifies 10 mutations overall. This greater resolution permitsfiner discrimination for males having the same genetic lineage, whichmay still provide random mutations despite a lack of recombination.

TABLE 4 Mutations found in the 53 father-son pairs for each multiplexpanel. Number of Marker mutations Yfiler ® Kit B Panel 7 DYF387S1^(a) 11 DYS449^(a) 2 2 DYS458 2 2 2 2 DYS518^(a) 3 3 DYS549 1 1 Y-GATA-H4 2 22 2 Total number of mutations/Kit 4 5 10 This marker is a rapidlymutating Y-STR marker.

Example 14

Discrimination of Male Samples of differing genetic backgrounds. Over700 male samples from a variety of genetic backgrounds are evaluatedusing Yfiler® and Panel 7 multiplex. The results are shown in TABLE 5a.While Yfiler® identifies a large percentage of individuals across a widerange of ethnic backgrounds; Panel 7 can identify a greater percentage,particularly in non-Caucasian groups.

TABLE 5a Discrimination of Male Samples of Differing GeneticBackgrounds. # of unique # of unique Total # male Yfiler ® Panel 7 Racesamples* haplotypes haplotypes African American 260 259 (99.6%) 260(100%)  Asian  5  5 (100%)  5 (100%) Caucasian 239 236 (98.7%) 239(100%)  Chinese  1 1 1 Hispanic  195* 193 (99.0%) 194 (99.5%) Korean 53*  52 (100%)  52 (100%) All  753* 741 (98.4%) 750 (99.6%)

of potentially related individuals removed

Further screening of male samples with Yfiler® identified a set of 15identical haplotypes. These identical haplotypes, including thepotentially related individuals from TABLE 5a were reevaluated with thePanel 7 multiplex and the results are shown in TABLE 5b. Seven of the 15identical Yfiler® haplotypes could be resolved. Four of the 15 identicalYfiler® haplotypes are potentially related; it is more difficult toresolve such haplotypes. Three of the identical Yfiler® haplotypescannot be resolved using Panel 7, even though the individuals arepotentially unrelated. However, Panel 7 multiplex can improve the rateof successfully resolving haplotype for individuals, including those ofgreater ethnic variety than previously seen in commercially availablemultiplex panels.

TABLE 5b Discrimination of Identical Haplotypes. Poten- tially No.Sample ID No. Race Panel 7 related 1 0590, 0976 Caucasian, Full matchHispanic (7/15 autosomal matches) 2 1153, 1175 2x Hispanic Full matchyes 3 0948, 0960 2x Hispanic Full match yes with 6 deletions 4 0403,0408 2x Caucasian 6 mismatches 5 0326, 0945 Caucasian, 5 mismatchesHispanic 6 0550, 0824, 0891 Caucasian, 2x 3 mismatches Hispanic resultedin 3 haplotypes 7 0627, 0507 2x Caucasian 1 mismatch at DYS481 (not arapidly mutating Y-STR) 8 0892, 0920 Hispanic, Full match Hispanic (7/15autosomal matches) 9 0497, 0601 2x Caucasian 6 mismatches 10 0011, 01632x African 2 mismatches at American DYS627 and DYS387S1ab 11 0242, 0591African A., 1 mismatch at Caucasian DYS449 12 0757, 0970 2x Hispanic TBD13 1077, 1082, 1091 3x Korean Full match yes 14 0781, 0869 2x HispanicFull match yes 15 1066, 1231 2x Korean Full match (12/15 autosomalmatches)

Example 15

Father-Son Study. Ninety five father-son pairs were studied, using boththe multiplex of Panel 7 and the multiplex of Y®filer. Of the ninetyfive pairs, nine pairs (9.5%) could be separated with the multiplex ofPanel 7 compared to 6 pairs (6.3%) that could be distinguished using themultiplex of Y®filer. TABLE 6 shows the particular marker which had adetectable mutation.

TABLE 6 Father-Son Pair Marker with Mutation Multiplex panel 52144_AF/C1DYS458 Panel 7, Y ®filer 52203_AF/C1 DYS458 Panel 7, Y ®filer DYS518Panel 7 52227_AF/C1 DYS576 Panel 7 52294_AF/C1 DYS448 Panel 7, Y ®filer52318_AF/C1 DYS448 Panel 7, Y ®filer 52361_AF/C1 DYS19 Panel 7, Y ®filer52369_AF/C1 DYS627 Panel 7 52142_AF/C1 SYD389II Panel 7, Y ®filer53160_AF/C1 DYF387S1ab Panel 7

What is claimed is:
 1. A method of amplifying alleles of Y-STR markersof a human male comprising the steps of: contacting a sample suspectedto contain a DNA sample of a human male with a set of multiplex analysisamplification primers comprising primers for the simultaneousamplification of the alleles of at least 27 Y-STR markers, wherein atleast 11 of the Y-STR markers are DYS576, DYS389I, DYS460, DYS458,DYS19, DYS456, DYS390, DYS570, DYS437, DYS393, and DYS439, wherein theamplification primers further comprise at least 5 Y-STR markers whichare rapidly mutating loci, wherein the at least 5 rapidly mutating Y-STRmarkers comprise DYF387S1ab, DYS449, DYS570, DYS576, and DYS627, whereinthe Y-STR markers beyond the 11 Y-STR markers have a base pair size lessthan about 410 base pairs, wherein at least one of the amplificationprimers comprises a mobility modifier; simultaneously amplifying thesample thereby forming a plurality of sets of amplicons of the at least11 Y-STR markers wherein each set of the amplicons has a base pair sizeless than 220 base pairs; and detecting each set of amplicons wherebythe alleles of the at least 11 Y-STR markers and the at least 5 rapidlymutating Y-STR markers are identified.
 2. The method of claim 1, whereinthe detecting is performed by separating the plurality of sets ofamplicons using a mobility dependent analysis.
 3. The method of claim 1,further comprising an additional set of primers for the amplification ofmore than the 11 Y-STR markers, wherein the additional set of primersare configured to provide sets of amplicons of the more than 11 Y-STRmarkers having a base pair size less than 410 base pairs.
 4. The methodof claim 1, wherein the 27 Y-STR markers are DYF387S1ab, DYS19,DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS460,DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS481, DYS518,DYS533, DYS570, DYS576, DYS627, DYS635, and Y-GATA-H4.
 5. The method ofclaim 1, wherein the 27 Y-STR markers are DYF387S1ab, DYS19, DYS385ab,DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS460, DYS437,DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS481, DYS533, DYS570,DYS576, DYS627, DYS635, DYS643, and Y-GATA-H4.
 6. The method of claim 2,wherein the mobility-dependent analytical technique is capillaryelectrophoresis.
 7. The method of claim 1, wherein the detecting isperformed by separating the plurality of sets of amplicons using amobility dependent analysis, wherein the plurality of sets of ampliconsare fluorescently labeled.
 8. The method of claim 1, wherein each set ofthe amplicons of the at least 11 Y-STR markers and the at least 5rapidly mutating Y-STR markers is labeled with one of at least 5 dyes.9. The method of claim 8, wherein the at least 5 dyes are fluorescentdyes configured to be spectrally distinct.
 10. The method of claim 1,wherein the mobility modifier is a non-nucleoside linker.
 11. The methodof claim 1, wherein the at least 5 rapidly mutating Y-STR markersfurther comprise DYS518.
 12. The method of claim 1, wherein the set ofprimers for the amplification of at least 11 Y-STR markers and the atleast 5 rapidly mutating Y-STR markers is a set of 25 primers for theamplification of DYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390,DYS391, DYS392, DYS393, DYS460, DYS437, DYS438, DYS439, DYS448, DYS449,DYS456, DYS458, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627, DYS635,and Y-GATA-H4.
 13. The method of claim 1, wherein the set of primers forthe amplification of at least 11 Y-STR markers and the at least 5rapidly mutating Y-STR markers is a set of 25 primers for theamplification of DYF387S1ab, DYS19, DYS385ab, DYS389I, DYS389II, DYS390,DYS391, DYS392, DYS393, DYS460, DYS437, DYS438, DYS439, DYS448, DYS449,DYS456, DYS458, DYS481, DYS533, DYS570, DYS576, DYS627, DYS635, DYS643,and Y-GATA-H4.
 14. The method of claim 1, performed using a kitcomprising: a) the primers of claim 1; and b) a size standard comprisingan allelic ladder.